bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence‐specific interference by small RNAs derived from adenovirus VAI RNA
Authors: Sano Masayuki , Kato Yoshio , Taira Kazunari
Published: February 2006

A virus‐associated RNA (VAI) of adenoviruses is a cytoplasmic non‐coding RNA and it plays an important role for viral replication in infected cells. VAI RNA transcripts, produced by RNA polymerase III (pol III), form tightly structured stems, which confer resistance to cellular defense systems. We demonstrate here that small RNAs of approximately 22 nucleotides are produced from a terminal stem region but not from an apical stem of VAI RNA. We determined the processing sites of VAI RNA by S1 nuclease mapping and further confirmed that the processed small RNA can act as small interfering RNAs (siRNAs) or as microRNAs (miRNAs) in transient transfection assays and during viral infection. Our data demonstrate that non‐coding RNAs synthesized by pol III can be substrates for Dicer, and diced small RNAs might regulate cellular phenomena as siRNAs and miRNAs.
Heat‐shock protein 27 is a major methylglyoxal‐modified protein in endothelial cells
Authors: Schalkwijk Casper G. , van Bezu Jan , van der Schors Roel C. , Uchida Koji , Stehouwer Coen D.A. , van Hinsbergh Victor W.M.
Published: February 2006

In endothelial cells cultured under high glucose conditions, methylglyoxal is the major intracellular precursor in the formation of advanced glycation endproducts. We found that endothelial cells incubated with 30 mM d‐glucose produced approximately 2‐fold higher levels of methylglyoxal but not 3‐deoxyglucosone and glyoxal, as compared to 5 mM d‐glucose. Under hyperglycaemic conditions, the methylglyoxal‐arginine adduct argpyrimidine as detected with a specific antibody, but not Ne‐(carboxymethyl)lysine and Ne‐(carboxyethyl)lysine, was significantly elevated. The glyoxylase I inhibitor HCCG and the PPARγ ligand troglitazone also increased argpyrimidine levels. Increased levels of argpyrimidine by glucose, HCCG and troglitazone are accompanied by a decrease in proliferation of endothelial cells. A 27 kDa protein was detected as a major argpyrimidine‐modified protein. With in‐gel digestion and mass spectrometric analysis, we identified this major protein as heat‐shock protein 27 (Hsp27). This argpyrimidine modification of Hsp27 may contribute to changes in endothelial cell function associated to diabetes.
Peptide substrate profiling defines fibroblast activation protein as an endopeptidase of strict Gly2‐Pro1‐cleaving specificity
Authors: Edosada Conrad Yap , Quan Clifford , Tran Thuy , Pham Victoria , Wiesmann Christian , Fairbrother Wayne , Wolf Beni B.
Published: February 2006

Fibroblast activation protein (FAP) is a serine protease of undefined endopeptidase specificity implicated in tumorigenesis. To characterize FAP's specificity, we synthesized intramolecularly quenched fluorescent substrate sets based on the FAP cleavage site in α2‐antiplasmin (TSGP‐NQ). FAP required substrates with Pro at P1 and Gly or d‐amino acids at P2 and preferred small, uncharged amino acids at P3, but tolerated most amino acids at P4, and . These substrate preferences allowed design of peptidyl‐chloromethyl ketones that inhibited FAP, but not the related protease, dipeptidyl peptidase‐4. Thus, FAP is a narrow specificity endopeptidase and this can be exploited for inhibitor design.
Deletions in the AL region of the h4xb plasma membrane Ca2+ pump
Authors: de Tezanos Pinto Felicitas , Adamo Hugo P.
Published: February 2006

Mutants of the plasma membrane Ca2+ pump (human isoform 4xb) with deletions in the linker between domain A and transmembrane segment M3 (AL region) were constructed and expressed in Chinese hamster ovary cells. The total or partial removal of the amino acid segment 300–349 did not change the maximal Ca2+ transport activity, but mutants with deletions involving residues 300–338 exhibited a higher apparent affinity for Ca2+ than the wild type h4xb enzyme. Deletion of the putative acidic lipid interacting sequence (residues 339–349) had no observable functional consequences. The removal of either residues 300–314 or 313–338 resulted in a similar increase in the apparent Ca2+ affinity of the pump although the increase was somewhat lower than that obtained by the deletion 300–349 suggesting that both deletions affected the same structural determinant. The results show that alterations in the region of the alternative splicing site A change the sensitivity to Ca2+ of the human isoform 4 of the PMCA.
Modulating the splicing activity of Tetrahymena ribozyme via RNA self‐assembly
Authors: Hasegawa Sumitaka , Rao Jianghong
Published: February 2006

The internal guiding sequence (IGS) is normally located at the 5′ end of trans‐splicing ribozymes that are derived from the Tetrahymena group I intron, and is required for the recognition of substrate RNAs and for trans‐splicing reactions. Here, we separated the Tetrahymena group I intron at the L2 loop to produce two fragments: the IGS‐containing substrate, and the IGS‐lacking ribozyme. We show here that two fragments can complex not through the IGS interaction but under the guidance of appended interacting nucleotides, and perform trans‐splicing. The splicing reactions took place both in vitro and in mammalian cells, and the spliced mRNA product from the self‐assembled ribozyme complex can be translated into functional proteins in vivo. The splicing efficiency was dependent on the length of appending nucleotides.
Metabolic cost of feeding in Atlantic Cod (Gadus morhua) larvae using microcalorimetry
Authors: McCollum Artie , Geubtner Jessica , Hunt von Herbing Ione
Published: February 2006

A microcalorimeter that measures total heat output (μW) was used to determine total metabolic rate (aerobic and anaerobic) and the cost of feeding (specific dynamic action, SDA) in larval Atlantic cod (Gadus morhua) from hatching to 4 weeks post-hatch at 10°C. Total heat output increased throughout development from 2.14 μW at first-feeding to 23.72 μW at 4 weeks post-hatch. SDA was determined by comparing the total heat output among unfed larvae and fed larvae simultaneously. Total heat output increased in the first 2 h after feeding with rotifers (Brachionus sp.) and Artemia, remained high for up to 10 h, was significantly higher in fed larvae than in unfed larvae, and ranged from 16.56 μW at first-feeding to 47.84 μW at 4 weeks post-hatch. The differences in total heat output between unfed and fed larvae were 14.42 μW and 24.12 μW, representing an increase in metabolic cost of feeding by a factor of 1.67 over the first 4 weeks of larval life. That the metabolic cost of feeding increased with development and remained elevated suggests that cod larvae allocate a large part of their energy budget to growth in order to meet the demands of their fast growth rates.
Worldwide status and perspective on gadoid culture
Authors: Rosenlund Grethe , Skretting Magnus
Published: February 2006

Currently, Atlantic cod (Gadus morhua) is the primary species being developed for commercial culture, with activities concentrated around the North Atlantic. In addition, closed life cycles have been established for haddock (Melanogrammus aeglefinus), pollock (Pollachius pollachius), and hake (Merluccius australis), but production of these species (in Canada, Spain, and Chile) is rather modest. In the short- to medium-term, Atlantic cod will be the dominant gadoid species in culture, and it is believed that production can reach levels similar to those of farmed salmon within the next 15–20 years. This development is possible because methods for year-round production of juveniles and significant hatchery capacity have been established. Also, there is a demand for farmed cod to fill the gap between increasing market needs and diminishing supply from fisheries. However, challenges must be met if cod farming is to reach its anticipated potential. Juvenile production must become more reliable in terms of survival and quality. For the on-growing phase, the supply of cost-efficient feeds produced from sustainable raw materials is of utmost importance. Consumer markets need to be developed with an emphasis on quality and food safety. Relatively little is known about health management for gadoid species.
Growth, survival, diet, and on-growing husbandry of haddock Melanogrammus aeglefinus in tanks and netpens
Authors: Treasurer James W. , Sveier Harald , Harvey Warren , Allen Roddy , Cutts Christopher J. , Mazorra de Quero Carlos , Ford Leslie
Published: February 2006

Growth of haddock (Melanogrammus aeglefinus) was assessed in onshore tanks in West Scotland. Fish were stocked at a mean weight of 15 g in July 2002, with a second stocking in October 2002 at 25 g. Fish had a mean weight of 755 ± 150 (s.d.) g at 20 months after stocking and were harvested. The specific growth rate of haddock was similar to Atlantic cod (Gadus morhua) during the first year but decreased by 20% after that. Liver biomass was 17.6% of the whole body weight, suggesting haddock had difficulty in utilizing dietary lipid. Reduced lipid levels in the diet had only a short-term effect on hepatosomatic index (HSI). Haddock that were held under 24-h light from the first summer solstice did not mature at an age of two years when compared with complete maturation of fish reared under ambient light. Mortality during the on-growing stage was high (28% of stock) and was attributed to Vibrio anguillarum infection, and possibly to enlarged livers. An assessment of quality found taste and texture to be as good or equal to wild North Sea haddock, and quality was improved with a five-day starvation period. Haddock mean weight, six months after transfer to netpens, was not significantly different from that of fish of the same age grown in tanks.
Maternal weight gain ranges for optimal fetal growth in Japanese women
Authors: Takimoto H. , Sugiyama T. , Fukuoka H. , Kato N. , Yoshiike N.
Published: February 2006

Objective: To identify adequate weight gain ranges during pregnancy in Japanese women. Method: Obstetric records from 2001 to 2002 for 46,659 term, singleton, vaginally delivered live births was used to estimate IUGR and macrosomia risk. Total maternal weight gain was grouped according to gestational age‐specific percentile values of weight gain as follows: “very low” (under the 25th), “low” (25th to 49th), “moderate” (50th to 74th), “high” (75th to 89th), and “very high” (90th and over). Results: About 6% of infants were identified as having IUGR and 0.9% as macrosomia. IUGR risk was elevated with low weight gains. Macrosomia risk was related to high weight gains and previous spontaneous abortions. Conclusion: Achieving weight gains between the 50th and 75th percentiles for gestational age was considered adequate for optimal fetal growth in Japanese pregnant women.
Timing of the late-glacial climate reversal in the Southern Hemisphere using high-resolution radiocarbon chronology for Kaipo Bog, New Zealand
Authors: Hajdas Irka , Lowe David J. , Newnham Rewi M. , Bonani Georges
Published: February 2006

The pattern of climate change in the Southern Hemisphere during the Younger Dryas (YD) chronozone provides essential constraint on mechanisms of abrupt climate change only if accurate, high-precision chronologies are obtained. A climate reversal reported previously at Kaipo bog, New Zealand, had been dated between 13,600 and 12,600 cal yr B.P. and appeared to asynchronously overlap the YD chron, but the chronology, based on conventionally radiocarbon-dated bulk sediment samples, left the precise timing questionable. We report a new high-resolution AMS 14C chronology for the Kaipo record that confirms the original chronology and provides further evidence for a mid-latitude Southern Ocean cooling event dated between 13,800 and 12,400 cal yr B.P. (2σ range), roughly equivalent to the Antarctic Cold Reversal.
Middle Weichselian environments on western Yamal Peninsula, Kara Sea based on pollen records
Authors: Andreev Andrei A. , Forman Steven L. , Ingólfsson Ólafur , Manley William F.
Published: February 2006

Pollen data from two sections from a coastal cliff on the western Yamal Peninsula (69°43.27′N, 66°48.80′E) document the environmental history during the Karginsky (Middle Weichselian) interstadial. Low pollen concentrations, high amounts of redeposited pollen, and relatively high presence of Artemisia pollen characterize sediments deposited at about 33,000 14C yr B.P. Grass-sedge plant associations with few other herbs occupied the area during the late Karginsky interstadial. Artemisia pollen may indicate rather xerophytic vegetation and disturbed soils in the area. The dominance of redeposited pollen reflects scarce (disturbed) vegetation cover and low pollen productivity. The climate was relatively cold and dry. Sediments dated to 32,400 14C yr B.P. contain fewer redeposited pollen and concentration of non-redeposited pollen is significantly higher. Pollen contents indicate the dominance of tundra-like grass-sedge vegetation and more humid conditions. Pollen records dated between 30,100 and 25,100 14C yr B.P. also reflect scarce tundra-like vegetation during this interval. The presence of Betula nana and Salix pollen may reflect limited presence of shrub communities. This suggests that the climate was somewhat warmer during the latter part of the interstadial. However, generally the pollen records show that harsh environmental conditions prevailed on the Yamal Peninsula during the Karginsky interstadial.
Schizophrenia, antipsychotics and diabetes: Genetic aspects
Authors: Bellivier F.
Published: February 2006

The relatively high comorbidity of type 2 diabetes and schizophrenia may suggest a shared biological susceptibility to these twoconditions. Family studies have demonstrated an increased risk of diabetes in unaffected relatives of patients with schizophrenia, consistent with a heritable susceptibility trait. Linkage analyses have identified several loci that are associated with schizophrenia and some of these, notably those on chromosomes 2p22.1-p13.2 and 6g21-824.1 have also been observed in linkage studies in type 2 diabetes. In addition, the dopamine D5 receptor on chromosome 5 and the tyrosine hydroxylase gene on chromosome 11 have both been suggested as candidate genes in schizophrenia and may also be implicated in susceptibility to poor glycaemic control. In addition, an increased rate of type II diabetes has been observed in some patients treated with antipsychotics. Potential neurochemical substrates of this effect include the histamine H1 receptor, the 5-HT2C serotonin receptor or the β3 adrenoreceptor. However, the search for a genetic basis to the association between diabetes and schizophrenia is still in its infancy, and much further work needs to be performed, including the systematic screening of all confirmed susceptibility loci and quantitative trait locus mapping of glycaemic control.
Antipsychotics and the risk of diabetes: A general data review
Authors: Bottai T. , Quintin P. , Perrin E.
Published: February 2006

Recently, attention has focused on a potential link between schizophrenia and diabetes, with speculation that this potential associationis stronger in patients who are prescribed atypical antipsychotics. Pharmacoepidemiological studies can help to evaluate this potential association. Source data on the incidence of diabetes in patients treated with antipsychotics is available in the FDA MedWatch database, prescription claims databases and other patient registries. These data indicate that antipsychotic drugs may increase the risk of developing diabetes and that there may be an interaction with age. However, current data are insufficient to accurately assess potential differences in the risk of diabetes between users of individual antipsychotic medications. In addition, antipsychotic treatment-emergent diabetes has several distinct features, notably relating to age of onset, gender ratio, rate of deterioration of glycaemic control, and independence from initial treatment emergent weight gain. Nonetheless, guidelines for the control of risk factors for diabetes developed for the general population appear to be applicable to patients with schizophrenia.
Are there specific care requirements for patients with schizophrenia and diabetes or with a risk of diabetes?
Authors: Thomas P. , Raymondet P. , Charbonnel B. , Vaiva G.
Published: February 2006

An interactive workshop was held to discuss the risk of diabetes in patients with schizophrenia, to evaluate the available data concerninghow such patients should be managed in terms of minimising the risk of diabetes and of optimising their care where diabetes to develop. The subjects discussed covered monitoring of risk factors, education about lifestyle and the risk of diabetes, patient care and treatment options, and interfaces between psychiatry and diabetology. The workshop noted that all patients with a diagnosis of schizophrenia had an elevated risk of developing diabetes and that this needed to be reflected in the follow-up of the patients in order to reduce the chances of the emergence of disease. The risk of diabetes is complicated by the presence of other risk factors and the intensity of the diabetes prevention programme needs to reflect adequately the overall risk. The most important prevention methods relate to lifestyle changes that patients with schizophrenia may be spontaneously unlikely to adopt and therefore necessitate the implementation of specific education measures aimed at patients and their families. The workshop proposed follow-up intensities, monitoring procedures and prevention programmes stratified according to the risk of developing diabetes. It was not considered that there was sufficient data available to orientate treatment choices between individual antipsychotic drugs according to the potential risk of developing diabetes.
A genetic locus required for iron acquisition in
Mycobacterium tuberculosis
Authors: Krithika R. , Marathe Uttara , Saxena Priti , Ansari Mohd. Zeeshan , Mohanty Debasisa , Gokhale Rajesh S.
Published: February 2006

Mycobactins are a family of membrane-associated siderophores required for
Mycobacterium tuberculosis
to adapt to its intracellular habitat. These lipophilic siderophores have been recently shown to directly acquire intracellular iron through lipid trafficking. Despite tremendous progress in understanding the assembly-line enzymology of the siderophore biosynthesis, the genes as well as the mechanistic and biochemical principles involved in producing membrane-associated siderophores have not been investigated. Here, we report a biosynthetic locus that incorporates variety of aliphatic chains on the mycobactin skeleton. Cell-free reconstitution studies demonstrate that these acyl chains are directly transferred from a carrier protein on to the ε-amino group of lysine residue by an unidentified Rv1347c gene product. The unsaturation in the lipidic chain is produced by a novel acyl-acyl carrier protein dehydrogenase, which, in contrast to the conventional acyl-CoA dehydrogenases, is involved in the biosynthetic pathway. MbtG protein then performs the final N6-hydroxylation step. Genome-wide analysis revealed homologues of
N
-acyl transferase and MbtG in other pathogenic bacteria. Because iron plays a key role in the development of infectious diseases, the biosynthetic pathway described here represents an attractive target for developing new antibacterial agents.
Mutation and genomic deletion status of
ataxia telangiectasia mutated
(
ATM
) and
p53
confer specific gene expression profiles in mantle cell lymphoma
Authors: Greiner Timothy C. , Dasgupta Chiranjib , Ho Vincent V. , Weisenburger Dennis D. , Smith Lynette M. , Lynch James C. , Vose Julie M. , Fu Kai , Armitage James O. , Braziel Rita M. , Campo Elias , Delabie Jan , Gascoyne Randy D. , Jaffe Elaine S. , Muller-Hermelink Hans K. , Ott German , Rosenwald Andreas , Staudt Louis M. , Im Michael Y. , Karaman Mazen W. , Pike Brian L. , Chan Wing C. , Hacia Joseph G.
Published: February 2006

Although mantle cell lymphoma (MCL) frequently harbors inactivated
ataxia telangiectasia mutated
(
ATM
) and
p53
alleles, little is known about the molecular phenotypes caused by these genetic changes. We identified point mutations and genomic deletions in these genes in a series of cyclin D1-positive MCL cases and correlated genotype with gene expression profiles and overall survival. Mutated and/or deleted
ATM
and
p53
alleles were found in 56% (40/72) and 26% (21/82) of the cases examined, respectively. Although MCL patients with inactive
p53
alleles showed a significant reduction in median overall survival, aberrant
ATM
status did not predict for survival. Nevertheless, specific gene expression signatures indicative of the mutation and genomic deletion status of each gene were identified that were different from wild-type cases. These signatures were comprised of a select group of genes related to apoptosis, stress responses, and cell cycle regulation that are relevant to
ATM
or
p53
function. Importantly, we found the molecular signatures are different between cases with mutations and deletions, because the latter are characterized by loss of genes colocalized in the same chromosome region of
ATM
or
p53
. This information on molecular phenotypes may provide new areas of investigation for
ATM
function or may be exploited by designing specific therapies for MCL cases with
p53
aberrations.
A microRNA expression signature of human solid tumors defines cancer gene targets
Authors: Volinia Stefano , Calin George A. , Liu Chang-Gong , Ambs Stefan , Cimmino Amelia , Petrocca Fabio , Visone Rosa , Iorio Marilena , Roldo Claudia , Ferracin Manuela , Prueitt Robyn L. , Yanaihara Nozumu , Lanza Giovanni , Scarpa Aldo , Vecchione Andrea , Negrini Massimo , Harris Curtis C. , Croce Carlo M.
Published: February 2006

Small noncoding microRNAs (miRNAs) can contribute to cancer development and progression and are differentially expressed in normal tissues and cancers. From a large-scale miRnome analysis on 540 samples including lung, breast, stomach, prostate, colon, and pancreatic tumors, we identified a solid cancer miRNA signature composed by a large portion of overexpressed miRNAs. Among these miRNAs are some with well characterized cancer association, such as
miR-17-5p
,
miR-20a
,
miR-21
,
miR-92
,
miR-106a
, and
miR-155
. The predicted targets for the differentially expressed miRNAs are significantly enriched for protein-coding tumor suppressors and oncogenes (
P
< 0.0001). A number of the predicted targets, including the tumor suppressors RB1 (Retinoblastoma 1) and TGFBR2 (transforming growth factor, beta receptor II) genes were confirmed experimentally. Our results indicate that miRNAs are extensively involved in cancer pathogenesis of solid tumors and support their function as either dominant or recessive cancer genes.
Antibodies to native myelin oligodendrocyte glycoprotein are serologic markers of early inflammation in multiple sclerosis
Authors: Lalive Patrice H. , Menge Til , Delarasse Cecile , Della Gaspera Bruno , Pham-Dinh Danielle , Villoslada Pablo , von Büdingen H.-C. , Genain Claude P.
Published: February 2006

Myelin oligodendrocyte glycoprotein (MOG) is an integral membrane protein expressed in CNS oligodendrocytes and outermost myelin lamellae. Anti-MOG Abs cause myelin destruction (demyelination) in animal models of multiple sclerosis (MS); however, such pathogenic Abs have not yet been characterized in humans. Here, a method that specifically detects IgG binding to human MOG in its native, membrane-embedded conformation on MOG-transfected mammalian cells was used to evaluate the significance of these auto Abs. Compared with healthy controls, native MOG-specific IgGs were most frequently found in serum of clinically isolated syndromes (P< 0.001) and relapsing-remitting MS (P< 0.01), only marginally in secondary progressive MS (P< 0.05), and not at all in primary progressive MS. We demonstrate that epitopes exposed in this cell-based assay are different from those exposed on the refolded, extracellular domain of human recombinant MOG tested by solid-phase ELISA. In marmoset monkeys induced to develop MS-like CNS inflammatory demyelination, IgG reactivity against the native membrane-bound MOG is always detected before clinical onset of disease (P< 0.0001), unlike that against other myelin constituents. We conclude that (i) epitopes displayed on native, glycosylated MOG expressedin vivoare early targets for pathogenic Abs; (ii) these Abs, which are not detected in solid-phase assays, might be the ones to play a pathogenic role in early MS with predominant inflammatory activity; and (iii) the cell-based assay provides a practical serologic marker for early detection of CNS autoimmune demyelination including its preclinical stage at least in the primate MS model.
Regulation of allergic airway inflammation through Toll-like receptor 4-mediated modification of mast cell function
Authors: Nigo Yukiko I. , Yamashita Masakatsu , Hirahara Kiyoshi , Shinnakasu Ryo , Inami Masamichi , Kimura Motoko , Hasegawa Akihiro , Kohno Yoichi , Nakayama Toshinori
Published: February 2006

In a mouse experimental asthma model, the administration of bacterial lipopolysaccharide (LPS), particularly at low doses, enhances the levels of ovalbumin (OVA)-induced eosinophilic airway inflammation. In an effort to clarify the cellular and molecular basis for the LPS effect, we demonstrate that the OVA-induced eosinophilic inflammation in the lung is dramatically increased by the administration of LPS in wild-type mice, whereas such increase was not observed in mast-cell-deficient mice or Toll-like receptor (TLR)4-deficient mice. Adoptive transfer of bone-marrow-derived mast cells (BMMCs) from wild-type, but not from TLR4-deficient, mice restored the increased eosinophilic inflammation in mast-cell-deficient mice. Wild-type BMMCs pretreated with LPSin vitroalso reconstituted the eosinophilic inflammation. Moreover,in vitroanalysis revealed that the treatment of BMMCs with LPS resulted in NF-κB activation, sustained up-regulation of GATA1 and -2 expression, and increased the capability to produce IL-5 and -13. Dramatic increases in the expression of IL-5 and -13 and Eotaxin 2 were detected in LPS-treated BMMCs after costimulation with LPS and IgE/Ag. Overexpression of GATA1, but not GATA2, in MC9 mast cells resulted in increased transcriptional activity of IL-4, -5, and -13. Furthermore, the levels of transcription of Th2 cytokines in BMMCs were decreased by the introduction of small interfering RNA for GATA1. Thus, mast cells appear to control allergic airway inflammation after their activation and modulation through TLR4-mediated induction of GATA1 and subsequent increase in Th2 cytokine production.
Gain-of-function/Noonan syndrome SHP-2/
Ptpn11
mutants enhance calcium oscillations and impair NFAT signaling
Authors: Uhlén Per , Burch Peter M. , Zito Christina Ivins , Estrada Manuel , Ehrlich Barbara E. , Bennett Anton M.
Published: February 2006

Gain-of-function mutations in SHP-2/
PTPN11
cause Noonan syndrome, a human developmental disorder. Noonan syndrome is characterized by proportionate short stature, facial dysmorphia, increased risk of leukemia, and congenital heart defects in ≈50% of cases. Congenital heart abnormalities are common in Noonan syndrome, but the signaling pathway(s) linking gain-of-function SHP-2 mutants to heart disease is unclear. Diverse cell types coordinate cardiac morphogenesis, which is regulated by calcium (Ca
2+
) and the nuclear factor of activated T-cells (NFAT). It has been shown that the frequency of Ca
2+
oscillations regulates NFAT activity. Here, we show that in fibroblasts, Ca
2+
oscillations in response to FGF-2 require the phosphatase activity of SHP-2. Conversely, gain-of-function mutants of SHP-2 enhanced FGF-2-mediated Ca
2+
oscillations in fibroblasts and spontaneous Ca
2+
oscillations in cardiomyocytes. The enhanced frequency of cardiomyocyte Ca
2+
oscillations induced by a gain-of-function SHP-2 mutant correlated with reduced nuclear translocation and transcriptional activity of NFAT. These data imply that gain-of-function SHP-2 mutants disrupt the Ca
2+
oscillatory control of NFAT, suggesting a potential mechanism for congenital heart defects in Noonan syndrome.
A pilot clinical trial of a recombinant ricin vaccine in normal humans
Authors: Vitetta Ellen S. , Smallshaw Joan E. , Coleman Elaine , Jafri Hasan , Foster Callie , Munford Robert , Schindler John
Published: February 2006

Ricin, a highly potent toxin produced by castor beans, is classified by the Centers for Disease Control and Prevention as a level B biothreat because it is easily produced, readily available, and highly stable. There have been >750 cases of documented ricin intoxication in humans. There is no approved vaccine for ricin. Ricin contains a lectin-binding B chain and a ribotoxic A chain (RTA). In addition to its ribotoxic site, we have identified a separate site on RTA that is responsible for inducing vascular leak syndrome (VLS) in humans. We have generated a recombinant RTA with two amino acid substitutions that disrupt its ribotoxic site (Y80A) and its VLS-inducing site (V76M). This mutant recombinant RTA (named RiVax) was expressed and produced in
Escherichia coli
and purified. When RiVax was injected i.m. into mice it protected them against a ricin challenge of 10 LD
50
s. Preclinical studies in both mice and rabbits demonstrated that RiVax was safe. Based on these results, we have now conducted a pilot clinical trial in humans under an investigational new drug application submitted to the Food and Drug Administration. In this study, three groups of five normal volunteers were injected three times at monthly intervals with 10, 33, or 100 μg of RiVax. The vaccine was safe and elicited ricin-neutralizing Abs in one of five individuals in the low-dose group, four of five in the intermediate-dose group, and five of five in the high-dose group. These results justify further development of the vaccine.
Structure and function of the SWIRM domain, a conserved protein module found in chromatin regulatory complexes
Authors: Da Guoping , Lenkart Jeffrey , Zhao Kehao , Shiekhattar Ramin , Cairns Bradley R. , Marmorstein Ronen
Published: February 2006

The SWIRM domain is a module found in the Swi3 and Rsc8 subunits of SWI/SNF-family chromatin remodeling complexes, and the Ada2 and BHC110/LSD1 subunits of chromatin modification complexes. Here we report the high-resolution crystal structure of the SWIRM domain from Swi3 and characterize the
in vitro
and
in vivo
function of the SWIRM domains from
Saccharomyces cerevisiae
Swi3 and Rsc8. The Swi3 SWIRM forms a four-helix bundle containing a pseudo 2-fold axis and a helix–turn–helix motif commonly found in DNA-binding proteins. We show that the Swi3 SWIRM binds free DNA and mononucleosomes with high and comparable affinity and that a subset of Swi3 substitution mutants that display growth defects
in vivo
also show impaired DNA-binding activity
in vitro
, consistent with a nucleosome targeting function of this domain. Genetic and biochemical studies also reveal that the Rsc8 and Swi3 SWIRM domains are essential for the proper assembly and
in vivo
functions of their respective complexes. Together, these studies identify the SWIRM domain as an essential multifunctional module for the regulation of gene expression.
“Genome design” model: Evidence from conserved intronic sequence in human–mouse comparison
Authors: Vinogradov Alexander E.
Published: February 2006

Introns are shorter in housekeeping genes than in tissue- or development-specific genes. Differing explanations have been offered for this phenomenon: selection for economy (in housekeeping genes), mutation bias or “genomic design.” The large-scale implementation in this present paper of a rigorous local sequence alignment algorithm revealed an unprecedented fraction of evolutionarily conserved DNA in human–mouse introns (∼60% of human and ∼70% of mouse intron length remained after masking for lineage-specific repeats). The length distributions of both conserved and nonconserved regions are very broad but show peaks close to nucleosomal and dinucleosomal DNA. Both the fraction of conserved sequence and its absolute length were higher in introns of tissue-specific genes than housekeeping genes. This difference remained after control for between-species identity of the conserved fraction, mutation rate, and GC content. In a more direct control, the product of the conserved sequence fraction and the between-species identity of this fraction (which can be considered to be the fraction of conserved nucleotides) was greater in introns of tissue-specific genes than housekeeping genes. Neither the fraction of intron length covered by repeats nor the balance of small insertions and deletions (indels) can explain the greater length of introns in tissue-specific genes. The length of the conserved intronic DNA in a gene is correlated with the number of functional domains in the protein encoded by that gene. These results suggest that the greater length of introns in tissue-specific genes is not due to selection for economy or mutation bias but instead is related to functional complexity (probably mediated by chromatin condensation), and that the evolution of the bulk of noncoding DNA is not completely neutral.
Utilization of a whole genome SNP panel for efficient genetic mapping in the mouse
Authors: Moran Jennifer L. , Bolton Andrew D. , Tran Pamela V. , Brown Alison , Dwyer Noelle D. , Manning Danielle K. , Bjork Bryan C. , Li Cheng , Montgomery Kate , Siepka Sandra M. , Vitaterna Martha Hotz , Takahashi Joseph S. , Wiltshire Tim , Kwiatkowski David J. , Kucherlapati Raju , Beier David R.
Published: February 2006

Phenotype-driven genetics can be used to create mouse models of human disease and birth defects. However, the utility of these mutant models is limited without identification of the causal gene. To facilitate genetic mapping, we developed a fixed single nucleotide polymorphism (SNP) panel of 394 SNPs as an alternative to analyses using simple sequence length polymorphism (SSLP) marker mapping. With the SNP panel, chromosomal locations for 22 monogenic mutants were identified. The average number of affected progeny genotyped for mapped monogenic mutations is nine. Map locations for several mutants have been obtained with as few as four affected progeny. The average size of genetic intervals obtained for these mutants is 43 Mb, with a range of 17–83 Mb. Thus, our SNP panel allows for identification of moderate resolution map position with small numbers of mice in a high-throughput manner. Importantly, the panel is suitable for mapping crosses from many inbred and wild-derived inbred strain combinations. The chromosomal localizations obtained with the SNP panel allow one to quickly distinguish between potentially novel loci or remutations in known genes, and facilitates fine mapping and positional cloning. By using this approach, we identified DNA sequence changes in two ethylnitrosourea-induced mutants.
Cholic Acid, a Bile Acid Elicitor of Hypersensitive Cell Death, Pathogenesis-Related Protein Synthesis, and Phytoalexin Accumulation in Rice
Authors: Koga Jinichiro , Kubota Hidetoshi , Gomi Shuichi , Umemura Kenji , Ohnishi Masao , Kono Toshiaki
Published: February 2006

When plants interact with certain pathogens, they protect themselves by generating various defense responses. These defense responses are induced by molecules called elicitors. Since long ago, composts fermented by animal feces have been used as a fertilizer in plant cultivation, and recently, have been known to provide suppression of plant disease. Therefore, we hypothesized that the compounds from animal feces may function as elicitors of plant defense responses. As a result of examination of our hypothesis, an elicitor of rice defense responses was isolated from human feces, and its structure was identified as cholic acid (CA), a primary bile acid in animals. Treatment of rice (Oryza sativa) leaves with CA induced the accumulation of antimicrobial compounds (phytoalexins), hypersensitive cell death, pathogenesis-related (PR) protein synthesis, and increased resistance to subsequent infection by virulent pathogens. CA induced these defense responses more rapidly than did fungal cerebroside, a sphingolipid elicitor isolated from the rice pathogenic fungus Magnaporthe grisea. Furthermore, fungal cerebroside induced both types of rice phytoalexins, phytocassanes and momilactones, whereas CA mainly induced phytocassanes, but not momilactones. In the structure-activity relationship analysis, the hydroxyl groups at C-7 and C-12, and the carboxyl group at C-24 of CA contributed to the elicitor activity. These results indicate that CA is specifically recognized by rice and is a different type of elicitor from fungal cerebroside. This report demonstrated that bile acid induced defense responses in plants.
A Role for Auxin Response Factor 19 in Auxin and Ethylene Signaling in Arabidopsis
Authors: Li Jisheng , Dai Xinhua , Zhao Yunde
Published: February 2006

Although auxin response factors (ARFs) are the first well-characterized proteins that bind to the auxin response elements, elucidation of the roles of each ARF gene in auxin responses and plant development has been challenging. Here we show that ARF19 and ARF7 not only participate in auxin signaling, but also play a critical role in ethylene responses in Arabidopsis (Arabidopsisthaliana) roots, indicating that the ARFs serve as a cross talk point between the two hormones. Both arf19 and arf7 mutants isolated from our forward genetic screens are auxin resistant and the arf19arf7 double mutant had stronger auxin resistance than the single mutants and displayed phenotypes not seen in the single mutants. Furthermore, we show that a genomic fragment of ARF19 not only complements arf19, but also rescues arf7. We conclude that ARF19 complements ARF7 at the protein level and that the ARF7 target sequences are also recognized by ARF19. Therefore, it is the differences in expression level/pattern and not the differences in protein sequences between the two ARFs that determines the relative contribution of the two ARFs in auxin signaling and plant development. In addition to being auxin resistant, arf19 has also ethylene-insensitive roots and ARF19 expression is induced by ethylene treatment. This work provides a sensitive genetic screen for uncovering auxin-resistant mutants including the described arf mutants. This study also provides a likely mechanism for coordination and integration of hormonal signals to regulate plant growth and development.
Point Mutations with Positive Selection Were a Major Force during the Evolution of a Receptor-Kinase Resistance Gene Family of Rice
Authors: Sun Xinli , Cao Yinglong , Wang Shiping
Published: February 2006

The rice (Oryza sativa) Xa26 gene, which confers resistance to bacterial blight disease and encodes a leucine-rich repeat (LRR) receptor kinase, resides at a locus clustered with tandem homologous genes. To investigate the evolution of this family, four haplotypes from the two subspecies of rice, indica and japonica, were analyzed. Comparative sequence analysis of 34 genes of 10 types of paralogs of the family revealed haplotype polymorphisms and pronounced paralog diversity. The orthologs in different haplotypes were more similar than the paralogs in the same haplotype. At least five types of paralogs were formed before the separation of indica and japonica subspecies. Only 7% of amino acid sites were detected to be under positive selection, which occurred in the extracytoplasmic domain. Approximately 74% of the positively selected sites were solvent-exposed amino acid residues of the LRR domain that have been proposed to be involved in pathogen recognition, and 73% of the hypervariable sites detected in the LRR domain were subject to positive selection. The family is formed by tandem duplication followed by diversification through recombination, deletion, and point mutation. Most variation among genes in the family is caused by point mutations and positive selection.
Silencing of a Germin-Like Gene in Nicotiana attenuata Improves Performance of Native Herbivores
Authors: Lou Yonggen , Baldwin Ian T.
Published: February 2006

Germins and germin-like proteins (GLPs) are known to function in pathogen resistance, but their involvement in defense against insect herbivores is poorly understood. In the native tobacco Nicotiana attenuata, attack from the specialist herbivore Manduca sexta or elicitation by adding larval oral secretions (OS) to wounds up-regulates transcripts of a GLP. To understand the function of this gene, which occurs as a single copy, we cloned the full-length NaGLP and silenced its expression in N. attenuata by expressing a 250-bp fragment in an antisense orientation with an Agrobacterium-based transformation system and by virus-induced gene silencing (VIGS). Homozygous lines harboring a single insert and VIGS plants had significantly reduced constitutive (measured in roots) and elicited NaGLP transcript levels (in leaves). Silencing NaGLP improved M. sexta larval performance and Tupiocoris notatus preference, two native herbivores of N. attenuata. Silencing NaGLP also attenuated the OS-induced hydrogen peroxide (H2O2), diterpene glycosides, and trypsin proteinase inhibitor responses, which may explain the observed susceptibility of antisense or VIGS plants to herbivore attack and increased nicotine contents, but did not influence the OS-elicited jasmonate and salicylate bursts, or the release of the volatile organic compounds (limonene, cis-α-bergamotene, and germacrene-A) that function as an indirect defense. This suggests that NaGLP is involved in H2O2 production and might also be related to ethylene production and/or perception, which in turn influences the defense responses of N. attenuata via H2O2 and ethylene-signaling pathways.
Contribution of the Alternative Pathway to Respiration during Thermogenesis in Flowers of the Sacred Lotus
Authors: Watling Jennifer R. , Robinson Sharon A. , Seymour Roger S.
Published: February 2006

We report results from in vivo measurements, using oxygen isotope discrimination techniques, of fluxes through the alternative and cytochrome respiratory pathways in thermogenic plant tissue, the floral receptacle of the sacred lotus (Nelumbo nucifera). Fluxes through both pathways were measured in thermoregulating flowers undergoing varying degrees of thermogenesis in response to ambient temperature. Significant increases in alternative pathway flux were found in lotus receptacles with temperatures 16°C to 20°C above ambient, but not in those with lesser amounts of heating. Alternative pathway flux in the hottest receptacles was 75% of the total respiratory flux. In contrast, fluxes through the cytochrome pathway did not change significantly during thermogenesis. These data support the hypothesis that increased flux through the alternative pathway is responsible for heating in the lotus and that it is unlikely that uncoupling proteins, which would have produced increased fluxes through the cytochrome pathway, contribute significantly to heating in this tissue. Comparisons of actual flux, with capacity determined using inhibitors, suggested that the alternative pathway was operating at close to maximum capacity in heating tissues of lotus. However, in nonheating tissues the inhibitor data significantly overestimated the alternative pathway flux. This confirms that isotopic measurements are necessary for accurate determination of fluxes through the two pathways.
The Geminivirus Nuclear Shuttle Protein NSP Inhibits the Activity ofAtNSI, a Vascular-Expressed Arabidopsis Acetyltransferase Regulated with the Sink-to-Source Transition
Authors: Carvalho Miguel F. , Turgeon Robert , Lazarowitz Sondra G.
Published: February 2006

DNA viruses can suppress or enhance the activity of cellular acetyltransferases to regulate virus gene expression and to affect cell cycle progression in support of virus replication. A role for protein acetylation in regulating the nuclear export of the bipartite geminivirus (Begomovirus) DNA genome was recently suggested by the findings that the viral movement protein NSP, a nuclear shuttle protein, interacts with the Arabidopsis (Arabidopsis thaliana) nuclear acetyltransferase AtNSI (nuclear shuttle protein interactor), and that this interaction and NSI expression are necessary for cabbage leaf curl virus infection and pathogenicity. To further investigate the consequences of NSI-NSP interactions, and the potential role of NSI in Arabidopsis growth and development, we used a reverse yeast two-hybrid selection and deletion analysis to identify NSI mutants that failed to interact with NSP, and promoter fusions to a uidA reporter gene to analyze the pattern of NSI expression during plant development. We found that NSI self assembles into highly active enzyme complexes and that high concentrations of NSP, in the absence of viral DNA, can inhibit NSI activity in vitro. Based on our detailed analysis of three NSI missense mutants, we identified an 88-amino acid putative domain, which spans NSI residues 107 to 194, as being required for both NSI oligomerization and its interaction with NSP. Finally, we found that NSI is predominantly transcribed in vascular cells, and that its expression is developmentally regulated in a manner that resembles the sink-to-source transition. Our data indicate that NSP can inhibit NSI activity by interfering with its assembly into highly active complexes, and suggest a mechanism by which NSP can both recruit NSI to regulate nuclear export of the viral genome and down-regulate NSI activity on cellular targets, perhaps to affect cellular differentiation and favor virus replication.
PLASTOCHRON2Regulates Leaf Initiation and Maturation in Rice
Authors: Kawakatsu Taiji , Itoh Jun-Ichi , Miyoshi Kazumaru , Kurata Nori , Alvarez Nena , Veit Bruce , Nagato Yasuo
Published: February 2006

In higher plants, leaves initiate in constant spatial and temporal patterns. Although the pattern of leaf initiation is a key element of plant shoot architecture, little is known about how the time interval between initiation events, termed plastochron, is regulated. Here, we present a detailed analysis of plastochron2 (pla2), a rice (Oryza sativa) mutant that exhibits shortened plastochron and precocious maturation of leaves during the vegetative phase and ectopic shoot formation during the reproductive phase. The corresponding PLA2 gene is revealed to be an orthologue of terminal ear1, a maize (Zea mays) gene that encodes a MEI2-like RNA binding protein. PLA2 is expressed predominantly in young leaf primordia. We show that PLA2 normally acts to retard the rate of leaf maturation but does so independently of PLA1, which encodes a member of the P450 family. Based on these analyses, we propose a model in which plastochron is determined by signals from immature leaves that act non-cell-autonomously in the shoot apical meristem to inhibit the initiation of new leaves.