bluuebunny/crossref_metadata_2025 huggingface dataset:
Engineering an artificial zymogen by alternate frame protein folding
Authors: Mitrea Diana M. , Parsons Lee S. , Loh Stewart N.
Published: January 2010

Alternate frame folding (AFF) is a novel mechanism by which allostery can be introduced into a protein where none may have existed previously. We employ this technology to convert the cytotoxic ribonuclease barnase into an artificial zymogen that is activated by HIV-1 protease. The AFF modification entails partial duplication of the polypeptide chain and mutation of a key catalytic residue in one of the duplicated segments. The resulting molecule can fold in one of two “frames” to yield the wild-type structure or a circularly permuted form in which the positions of the N- and C-termini are exchanged with a surface loop. It cannot take on both structures simultaneously because each competes for a shared amino acid sequence. An HIV-1 protease recognition sequence is inserted into one of the surface loops in the nonpermuted frame, and cleavage induces a shift from the nonpermuted fold to the permuted fold. Using the AFF mechanism, we were able to suppress
k
cat
/
K

M

by 250-fold in the proenzyme relative to wild-type barnase. HIV-1 protease cleavage subsequently increases
k
cat
/
K

M

by 130-fold. AFF is significant because it is general and can in principle be used to control activity of many enzymes, including those whose functions are not regulated by any existing mechanism.
Hsp90 inhibitors block outgrowth of EBV-infected malignant cells in vitro and in vivo through an EBNA1-dependent mechanism
Authors: Sun Xiaoping , Barlow Elizabeth A. , Ma Shidong , Hagemeier Stacy R. , Duellman Sarah J. , Burgess Richard R. , Tellam Judy , Khanna Rajiv , Kenney Shannon C.
Published: January 2010

EBV causes infectious mononucleosis and is associated with certain malignancies. EBV nuclear antigen 1 (EBNA1) mediates EBV genome replication, partition, and transcription, and is essential for persistence of the viral genome in host cells. Here we demonstrate that Hsp90 inhibitors decrease EBNA1 expression and translation, and that this effect requires the Gly-Ala repeat domain of EBNA1. Hsp90 inhibitors induce the death of established, EBV-transformed lymphoblastoid cell lines at doses nontoxic to normal cells, and this effect is substantially reversed when lymphoblastoid cell lines are stably infected with a retrovirus expressing a functional EBNA1 mutant lacking the Gly-Ala repeats. Hsp90 inhibitors prevent EBV transformation of primary B cells, and strongly inhibit the growth of EBV-induced lymphoproliferative disease in SCID mice. These results suggest that Hsp90 inhibitors may be particularly effective for treating EBV-induced diseases requiring the continued presence of the viral genome.
Reengineering orthogonally selective riboswitches
Authors: Dixon Neil , Duncan John N. , Geerlings Torsten , Dunstan Mark S. , McCarthy John E. G. , Leys David , Micklefield Jason
Published: January 2010

The ability to independently control the expression of multiple genes by addition of distinct small-molecule modulators has many applications from synthetic biology, functional genomics, pharmaceutical target validation, through to gene therapy. Riboswitches are relatively simple, small-molecule–dependent, protein-free, mRNA genetic switches that are attractive targets for reengineering in this context. Using a combination of chemical genetics and genetic selection, we have developed riboswitches that are selective for synthetic “nonnatural” small molecules and no longer respond to the natural intracellular ligands. The orthogonal selectivity of the riboswitches is also demonstrated in vitro using isothermal titration calorimetry and x-ray crystallography. The riboswitches allow highly responsive, dose-dependent, orthogonally selective, and dynamic control of gene expression in vivo. It is possible that this approach may be further developed to reengineer other natural riboswitches for application as small-molecule responsive genetic switches in both prokaryotes and eukaryotes.
Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals
Authors: Tizzano Marco , Gulbransen Brian D. , Vandenbeuch Aurelie , Clapp Tod R. , Herman Jake P. , Sibhatu Hiruy M. , Churchill Mair E. A. , Silver Wayne L. , Kinnamon Sue C. , Finger Thomas E.
Published: January 2010

The upper respiratory tract is continually assaulted with harmful dusts and xenobiotics carried on the incoming airstream. Detection of such irritants by the trigeminal nerve evokes protective reflexes, including sneezing, apnea, and local neurogenic inflammation of the mucosa. Although free intra-epithelial nerve endings can detect certain lipophilic irritants (e.g., mints, ammonia), the epithelium also houses a population of trigeminally innervated solitary chemosensory cells (SCCs) that express T2R bitter taste receptors along with their downstream signaling components. These SCCs have been postulated to enhance the chemoresponsive capabilities of the trigeminal irritant-detection system. Here we show that transduction by the intranasal solitary chemosensory cells is necessary to evoke trigeminally mediated reflex reactions to some irritants including acyl–homoserine lactone bacterial quorum-sensing molecules, which activate the downstream signaling effectors associated with bitter taste transduction. Isolated nasal chemosensory cells respond to the classic bitter ligand denatonium as well as to the bacterial signals by increasing intracellular Ca
2
+
. Furthermore, these same substances evoke changes in respiration indicative of trigeminal activation. Genetic ablation of either Gα-gustducin or TrpM5, essential elements of the T2R transduction cascade, eliminates the trigeminal response. Because acyl–homoserine lactones serve as quorum-sensing molecules for Gram-negative pathogenic bacteria, detection of these substances by airway chemoreceptors offers a means by which the airway epithelium may trigger an epithelial inflammatory response before the bacteria reach population densities capable of forming destructive biofilms.
ACAT1 gene ablation increases 24(S)-hydroxycholesterol content in the brain and ameliorates amyloid pathology in mice with AD
Authors: Bryleva Elena Y. , Rogers Maximillian A. , Chang Catherine C.Y. , Buen Floyd , Harris Brent T. , Rousselet Estelle , Seidah Nabil G. , Oddo Salvatore , LaFerla Frank M. , Spencer Thomas A. , Hickey William F. , Chang Ta-Yuan
Published: January 2010

Cholesterol metabolism has been implicated in the pathogenesis of several neurodegenerative diseases, including the abnormal accumulation of amyloid-β, one of the pathological hallmarks of Alzheimer disease (AD). Acyl-CoA:cholesterol acyltransferases (ACAT1 and ACAT2) are two enzymes that convert free cholesterol to cholesteryl esters. ACAT inhibitors have recently emerged as promising drug candidates for AD therapy. However, how ACAT inhibitors act in the brain has so far remained unclear. Here we show that ACAT1 is the major functional isoenzyme in the mouse brain. ACAT1 gene ablation
(
A1−) in triple transgenic (i.e., 3XTg-AD) mice leads to more than 60% reduction in full-length human APPswe as well as its proteolytic fragments, and ameliorates cognitive deficits. At 4 months of age, A1− causes a 32% content increase in 24-hydroxycholesterol (24SOH), the major oxysterol in the brain. It also causes a 65% protein content decrease in HMG-CoA reductase (HMGR) and a 28% decrease in sterol synthesis rate in AD mouse brains. In hippocampal neurons, A1− causes an increase in the 24SOH synthesis rate; treating hippocampal neuronal cells with 24SOH causes rapid declines in hAPP and in HMGR protein levels. A model is provided to explain our findings: in neurons, A1− causes increases in cholesterol and 24SOH contents in the endoplasmic reticulum, which cause reductions in hAPP and HMGR protein contents and lead to amelioration of amyloid pathology. Our study supports the potential of ACAT1 as a therapeutic target for treating certain forms of AD.
IL-10 directly suppresses CD4 but not CD8 T cell effector and memory responses following acute viral infection
Authors: Brooks David G. , Walsh Kevin B. , Elsaesser Heidi , Oldstone Michael B. A.
Published: January 2010

Mounting effective T cell responses is critical for eliciting long-lasting immunity following viral infection and vaccination. A multitude of inhibitory and stimulatory factors are induced following infection, and it is the compilation of these signals that quantitatively and qualitatively program the ensuing effector and memory T cell response. In response to lymphocytic choriomeningitis virus (LCMV) infection, the immunosuppressive cytokine IL-10 is rapidly up-regulated; however, how IL-10 is regulating what is often considered an “optimal” immune response is unclear. We demonstrate that IL-10 directly inhibits effector and memory CD4 T cell responses following an acutely resolved viral infection. Blockade of IL-10 enhanced the magnitude and the functional capacity of effector CD4 T cells that translated into increased and more effective memory responses. On the other hand, lack of IL-10 signaling did not impact memory CD8 T cell development. We propose that blockade of IL-10 may be an effective adjuvant to specifically enhance CD4 T cell immunity and protection following vaccination.
Agmatidine, a modified cytidine in the anticodon of archaeal tRNA
Ile
, base pairs with adenosine but not with guanosine
Authors: Mandal Debabrata , Köhrer Caroline , Su Dan , Russell Susan P. , Krivos Kady , Castleberry Colette M. , Blum Paul , Limbach Patrick A. , Söll Dieter , RajBhandary Uttam L.
Published: January 2010

Modification of the cytidine in the first anticodon position of the AUA decoding tRNA
Ile
(

) of bacteria and archaea is essential for this tRNA to read the isoleucine codon AUA and to differentiate between AUA and the methionine codon AUG. To identify the modified cytidine in archaea, we have purified this tRNA species from
Haloarcula marismortui
, established its codon reading properties, used liquid chromatography–mass spectrometry (LC-MS) to map RNase A and T1 digestion products onto the tRNA, and used LC-MS/MS to sequence the oligonucleotides in RNase A digests. These analyses revealed that the modification of cytidine in the anticodon of

adds 112 mass units to its molecular mass and makes the glycosidic bond unusually labile during mass spectral analyses. Accurate mass LC-MS and LC-MS/MS analysis of total nucleoside digests of the

demonstrated the absence in the modified cytidine of the C2-oxo group and its replacement by agmatine (decarboxy-arginine) through a secondary amine linkage. We propose the name agmatidine, abbreviation C
+
, for this modified cytidine. Agmatidine is also present in
Methanococcus maripaludis

and in
Sulfolobus solfataricus
total tRNA, indicating its probable occurrence in the AUA decoding tRNA
Ile
of euryarchaea and crenarchaea. The identification of agmatidine shows that bacteria and archaea have developed very similar strategies for reading the isoleucine codon AUA while discriminating against the methionine codon AUG.
Peptide vaccines prevent tumor growth by activating T cells that respond to native tumor antigens
Authors: Jordan Kimberly R. , McMahan Rachel H. , Kemmler Charles B. , Kappler John W. , Slansky Jill E.
Published: January 2010

Peptide vaccines enhance the response of T cells toward tumor antigens and represent a strategy to augment antigen-independent immunotherapies of cancer. However, peptide vaccines that include native tumor antigens rarely prevent tumor growth. We have assembled a set of peptide variants for a mouse-colon tumor model to determine how to improve T-cell responses. These peptides have similar affinity for MHC molecules, but differ in the affinity of the peptide-MHC/T-cell receptor interaction with a tumor-specific T-cell clone. We systematically demonstrated that effective antitumor responses are generated after vaccination with variant peptides that stimulate the largest proportion of endogenous T cells specific for the native tumor antigen. Importantly, we found some variant peptides that strongly stimulated a specific T-cell clone in vitro, but elicited fewer tumor-specific T cells in vivo, and were not protective. The T cells expanded by the effective vaccines responded to the wild-type antigen by making cytokines and killing target cells, whereas most of the T cells expanded by the ineffective vaccines only responded to the peptide variants. We conclude that peptide-variant vaccines are most effective when the peptides react with a large responsive part of the tumor-specific T-cell repertoire.
Targeted disruption of the CREB coactivator Crtc2 increases insulin sensitivity
Authors: Wang Yiguo , Inoue Hiroshi , Ravnskjaer Kim , Viste Kristin , Miller Nina , Liu Yi , Hedrick Susan , Vera Liliana , Montminy Marc
Published: January 2010

Under fasting conditions, increases in circulating concentrations of pancreatic glucagon maintain glucose homeostasis through induction of gluconeogenic genes by the CREB coactivator CRTC2. Hepatic CRTC2 activity is elevated in obesity, although the extent to which this cofactor contributes to attendant increases in insulin resistance is unclear. Here we show that mice with a knockout of the CRTC2 gene have decreased circulating glucose concentrations during fasting, due to attenuation of the gluconeogenic program. CRTC2 was found to stimulate hepatic gene expression in part through an N-terminal CREB binding domain that enhanced CREB occupancy over relevant promoters in response to glucagon. Deletion of sequences encoding the CREB binding domain in CRTC2
−/−
mice lowered circulating blood glucose concentrations and improved insulin sensitivity in the context of diet-induced obesity. Our results suggest that small molecules that attenuate the CREB–CRTC2 pathway may provide therapeutic benefit to individuals with type 2 diabetes.
Reintroducing Prediction to Explanation
Authors: Douglas Heather E.
Published: January 2010

Although prediction has been largely absent from discussions of explanation for the past 40 years, theories of explanation can gain much from a reintroduction. I review the history that divorced prediction from explanation, examine the proliferation of models of explanation that followed, and argue that accounts of explanation have been impoverished by the neglect of prediction. Instead of a revival of the symmetry thesis, I suggest that explanation should be understood as a cognitive tool that assists us in generating new predictions. This view of explanation and prediction clarifies what makes an explanation scientific and why inference to the best explanation makes sense in science.
Quantum Field Theory: Underdetermination, Inconsistency, and Idealization
Authors: Fraser Doreen
Published: January 2010

Quantum field theory (QFT) presents a genuine example of the underdetermination of theory by empirical evidence. There are variants of QFT—for example, the standard textbook formulation and the rigorous axiomatic formulation—that are empirically indistinguishable yet support different interpretations. This case is of particular interest to philosophers of physics because, before the philosophical work of interpreting QFT can proceed, the question of which variant should be subject to interpretation must be settled. New arguments are offered for basing the interpretation of QFT on a rigorous axiomatic variant of the theory. The pivotal considerations are the roles that consistency and idealization play in this case.
Reduction of RF-sheaths potentials by compensation or suppression of parallel RF currents on ICRF antennas
Authors: Mendes A. , Colas L. , Vulliez K. , Ekedahl A. , Argouarch A. , Milanesio D.
Published: January 2010

Radio frequency (RF) sheaths are suspected of limiting the performance of present-day ion cyclotron range of frequencies (ICRFs) antennas over long pulses and should be minimized in future fusion devices. Within the simplest models, RF-sheath effects are quantified by the integral V
RF = ∫ E
∥ · dl where the parallel RF field E
∥ is linked with the slow wave. On ‘long open field lines’ with large toroidal extension on both sides of the antenna it was shown that V
RF is excited by parallel RF currents j
∥ flowing on the antenna structure.

In this paper, the validity of this simple sheath theory is tested experimentally on the Tore Supra (TS) ITER-like antenna prototype (ILP), together with antenna simulation and post-processing codes developed to compute V
RF. The predicted poloidal localization of high-|V
RF| zones is confronted to that inferred from experimental data analysis. Surface temperature distribution on ILP front face, as well as ILP-induced modifications of RF coupling and hot spots on a magnetically connected lower hybrid current drive antenna, indicates local maxima of dc plasma potential in both the upper and lower parts of the ILP. This result, qualitatively conforming to V
RF simulations, is interpreted in terms of j
∥ flowing on ILP frame.

Once the validation is done, such reliable theoretical models and numerical codes are then employed to provide predictive results. Indeed, we propose two ways to reduce |V
RF| by acting on j
∥ on the antenna front face. The first method, more adapted for protruding antennas, consists of avoiding the j
∥ circulation on the antenna structure, by slotting the antenna frame on its horizontal edges and by partially cutting the Faraday screen rods.

The second method, well suited for recessed antennas, consists of compensating j
∥ of opposite signs along long flux tubes, with parallelepiped antennas aligned with (tilted) flux tubes.

The different concepts are assessed numerically on a two-strap TS antenna phased [0, π] using near RF fields from the antenna code TOPICA. Simulations stress the need to suppress all current paths for j
∥ to substantially reduce |V
RF| over the whole antenna height.
Tropomyosin and Myosin-II Cellular Levels Promote Actomyosin Ring Assembly in Fission Yeast
Authors: Stark Benjamin C. , Sladewski Thomas E. , Pollard Luther W. , Lord Matthew
Published: January 2010

Myosin-II (Myo2p) and tropomyosin are essential for contractile ring formation and cytokinesis in fission yeast. Here we used a combination of in vivo and in vitro approaches to understand how these proteins function at contractile rings. We find that ring assembly is delayed in Myo2p motor and tropomyosin mutants, but occurs prematurely in cells engineered to express two copies of myo2. Thus, the timing of ring assembly responds to changes in Myo2p cellular levels and motor activity, and the emergence of tropomyosin-bound actin filaments. Doubling Myo2p levels suppresses defects in ring assembly associated with a tropomyosin mutant, suggesting a role for tropomyosin in maximizing Myo2p function. Correspondingly, tropomyosin increases Myo2p actin affinity and ATPase activity and promotes Myo2p-driven actin filament gliding in motility assays. Tropomyosin achieves this by favoring the strong actin-bound state of Myo2p. This mode of regulation reflects a role for tropomyosin in specifying and stabilizing actomyosin interactions, which facilitates contractile ring assembly in the fission yeast system.
Muscle Biopsy and In Vitro Contracture Test in Subjects with Idiopathic HyperCKemia
Authors: Malandrini Alessandro , Orrico Alfredo , Gaudiano Carmen , Gambelli Simona , Galli Lucia , Berti Gianna , Tegazzin Vincenzo , Dotti Maria Teresa , Federico Antonio , Sorrentino Vincenzo
Published: January 2010

Background

Persistent high creatine kinase (CK) levels may reflect underlying subclinical myopathies. In most cases, pathogenesis is unknown and clinical management is unclear. Though clinically asymptomatic, these subjects are potentially susceptible to malignant hyperthermia.

Methods

The authors analyzed 37 subjects with persistent elevation of CK without significant weakness or other neurologic symptoms. Neurologic examination was performed according to manual muscle testing. Muscle biopsy and the in vitro contracture test were performed in all subjects.

Results

Twenty-three subjects (51.1%) were completely asymptomatic. The others had minor symptoms such as occasional cramps (11 subjects, 24.4%), fatigue (5 subjects, 11.1%), a combination of cramps and fatigue (5 subjects, 11.1%), and muscle pain (1 case, 2.2%). Muscle biopsy enabled precise diagnosis in 3 cases and was normal in 3 cases. The more frequent changes were variation in fiber size (31.1%), a combination of nuclear internalization and variation in fiber size (26.6%), nuclear internalization (6.6%), minor mitochondrial changes (4.4%), and neurogenic atrophy (4.4%). Immunocytochemical analysis was normal in all patients. In vitro contracture testing detected one malignant hyperthermia-susceptible and one malignant hyperthermia-equivocal subject.

Conclusions

The evidence of malignant hyperthermia susceptibility by in vitro contracture test seems to be relatively infrequent among subjects with idiopathic hyperCKemia, but the incidence of true malignant hyperthermia in idiopathic hyperCKemia is unknown. Muscle biopsy should be considered a useful, though not very sensitive, diagnostic tool in idiopathic hyperCKemia, because it enables potentially treatable disorders, such as inflammatory myopathies, to be discovered. No uniform morphologic finding typical of idiopathic hyperCKemia or malignant hyperthermia susceptibility was identified by muscle biopsy.
Brief Periods of Nitric Oxide Inhalation Protect against Myocardial Ischemia–Reperfusion Injury
Authors: Nagasaka Yasuko , Fernandez Bernadette O. , Garcia-Saura Maria F. , Petersen Bodil , Ichinose Fumito , Bloch Kenneth D. , Feelisch Martin , Zapol Warren M.
Published: January 2010

Background

Prolonged breathing of nitric oxide reduces myocardial ischemia-reperfusion injury, but the precise mechanisms responsible for the cardioprotective effects of inhaled nitric oxide are incompletely understood.

Methods

The authors investigated the fate of inhaled nitric oxide (80 parts per million) in mice and quantified the formation of nitric oxide metabolites in blood and tissues. The authors tested whether the accumulation of nitric oxide metabolites correlated with the ability of inhaled nitric oxide to protect against cardiac ischemia-reperfusion injury.

Results

Mice absorbed nitric oxide in a nearly linear fashion (0.19 +/- 0.02 micromol/g x h). Breathing nitric oxide rapidly increased a broad spectrum of nitric oxide metabolites. Levels of erythrocytic S-nitrosothiols, N-nitrosamines, and nitrosyl-hemes increased dramatically within 30 s of commencing nitric oxide inhalation. Marked increases of lung S-nitrosothiol and liver N-nitrosamine levels were measured, as well as elevated cardiac and brain nitric oxide metabolite levels. Breathing low oxygen concentrations potentiated the ability of inhaled nitric oxide to increase cardiac nitric oxide metabolite levels. Concentrations of each nitric oxide metabolite, except nitrate, rapidly reached a plateau and were similar after 5 and 60 min. In a murine cardiac ischemia-reperfusion injury model, breathing nitric oxide for either 5 or 60 min before reperfusion decreased myocardial infarction size as a fraction of myocardial area at risk by 31% or 32%, respectively.

Conclusions

Breathing nitric oxide leads to the rapid accumulation of a variety of nitric oxide metabolites in blood and tissues, contributing to the ability of brief periods of nitric oxide inhalation to provide cardioprotection against ischemia-reperfusion injury. The nitric oxide metabolite concentrations achieved in a target tissue may be more important than the absolute amounts of nitric oxide absorbed.
Biologic Effects of Nitrous Oxide
Authors: Sanders Robert D. , Weimann Jörg , Maze Mervyn , Warner David S. , Warner Mark A.
Published: January 2010

Nitrous oxide is the longest serving member of the anesthesiologist's pharmacologic armamentarium but remains a source of controversy because of fears over its adverse effects. Recently, the Evaluation of Nitrous oxide In a Gas Mixture for Anaesthesia (ENIGMA) trial reported that nitrous oxide use increases postoperative complications; further preclinical reports have suggested that nitrous oxide may contribute to neurocognitive dysfunction in the young and elderly. Therefore, nitrous oxide's longevity in anesthetic practice is under threat. In this article, the authors discuss the evidence for the putative toxicity of nitrous oxide, from either patient or occupational exposure, within the context of the mechanism of nitrous oxide's action. Although it would seem prudent to avoid nitrous oxide in certain vulnerable populations, current evidence in support of a more widespread prescription from clinical practice is unconvincing.
Comparison of the Immediate Effects of Surgical Incision on Dorsal Horn Neuronal Receptive Field Size and Responses during Postnatal Development
Authors: Ririe Douglas G. , Bremner Lindsay R. , Fitzgerald Maria
Published: January 2010

Background

Pain behavior in response to skin incision is developmentally regulated, but little is known about the underlying neuronal mechanisms. The authors hypothesize that the spatial activation and intensity of dorsal horn neuron responses to skin incision differ in immature and adult spinal cord.

Methods

Single wide-dynamic-range dorsal horn cell spike activity was recorded for a minimum of 2 h from anesthetized rat pups aged 7 and 28 days. Cutaneous pinch and brush receptive fields were mapped and von Frey hair thresholds were determined on the plantar hind paw before and 1 h after a skin incision was made.

Results

Baseline receptive field areas for brush and pinch were larger and von Frey thresholds lower in the younger animals. One hour after the incision, brush and pinch receptive field area, spontaneous firing, and evoked spike activity had significantly increased in the 7-day-old animals but not in the 28-day-old animals. Von Frey hair thresholds decreased at both ages.

Conclusions

Continuous recording from single dorsal horn cells both before and after injury shows that sensitization of receptive fields and of background and afferent-evoked spike activity at 1 h is greater in younger animals. This difference is not reflected in von Frey mechanical thresholds. These results highlight the importance of studying the effects of injury on sensory neuron physiology. Injury in young animals induces a marked and rapid increase in afferent-evoked activity in second-order sensory neurons, which may be important when considering long-term effects and analgesic interventions.
Secretion and translocation signals and DspB/F-binding domains in the type III effector DspA/E of Erwinia amylovora
Authors: Oh Chang-Sik , Carpenter Sara C. D. , Hayes Marshall L. , Beer Steven V.
Published: January 2010

DspA/E is a type III effector of Erwinia amylovora, the bacterial pathogen that causes fire blight disease in roseaceous plants. This effector is indispensable for disease development, and it is translocated into plant cells. A DspA/E-specific chaperone, DspB/F, is necessary for DspA/E secretion and possibly for its translocation. In this work, DspB/F-binding sites and secretion and translocation signals in the DspA/E protein were determined. Based on yeast two-hybrid assays, DspB/F was found to bind DspA/E within the first 210 amino acids of the protein. Surprisingly, both DspB/F and OrfA, the putative chaperone of Eop1, also interacted with the C-terminal 1059 amino acids of DspA/E; this suggests another chaperone-binding site. Secretion and translocation assays using serial N-terminal lengths of DspA/E fused with the active form of AvrRpt2 revealed that at least the first 109 amino acids, including the first N-terminal chaperone-binding motif and DspB/F, were required for efficient translocation of DspA/E, although the first 35 amino acids were sufficient for its secretion and the presence of DspB/F was not required. These results indicate that secretion and translocation signals are present in the N terminus of DspA/E, and that at least one DspB/F-binding motif is required for efficient translocation into plant cells.
Networks and dominance hierarchies: does interspecific aggression explain flower partitioning among stingless bees?
Authors: DWORSCHAK KAI , BLÜTHGEN NICO
Published: January 2010

1. The distribution of consumers among resources (trophic interaction network) may be shaped by asymmetric competition. Dominance hierarchy models predict that asymmetric interference competition leads to a domination of high quality resources by hierarchically superior species.

2. In order to determine the competitive dominance hierarchy and its effect on flower partitioning in a local stingless bee community in Borneo, interspecific aggressions were tested among eight species in arena experiments.

3. All species tested were strongly mutually aggressive in the arena, and the observed interactions were often lethal for one or both opponents. Aggression significantly increased with body size differences between fighting pairs and was asymmetric: larger aggressors were superior over smaller species. Additional aggression tests involved dummies with surface extracts, and results suggest that species‐ and colony‐specific surface profiles are important in triggering the aggressive behaviour.

4. Sixteen stingless bee species were observed foraging on 41 species of flowering plants. The resulting bee–flower interaction network showed a high degree of generalisation (network‐level specialisation H2’ = 0.11), corresponding to a random, opportunistic distribution of bee species among available flower species.

5. Aggressions on flowers were rare and only occurred at a low level. The dominance hierarchy obtained in the arena experiments did not correlate significantly with plant quality, estimated as the number of flowers per plant or as total bee visitation rate.

6. Our findings suggest that asymmetries in interference competition do not necessarily translate into actual resource partitioning in the context of complex interacting communities.
Embryo growth rates in birds and mammals
Authors: Ricklefs Robert E.
Published: January 2010

1. Embryo mass growth curves of 36 species of bird and 18 species of mammal were fitted by sigmoidal Gompertz functions, in whichk(days−1) describes the rate at which the embryo approaches an asymptotic massA(g).

2. The parameters of the Gompertz function were uncorrelated with parameters of power functions fitted to the same growth data, indicating that the two models describe different aspects of growth.

3. Asymptotes of the Gompertz functions for embryonic growth averaged 2·5 times neonate size, but were well below adult mass. Thus, the pre‐ and postnatal phases of growth are distinct and have different target sizes.

4. Embryo growth rate (k) decreases as the −1/4 power of neonate size; values for mammals were 63% of those of birds, on average.

5. Embryo growth rate can be predicted solely by the length of the incubation or gestation period, regardless of the size of the neonate, with the same relationship for birds and mammals.

6. Postnatal growth rate (kG) scales linearly with embryonic growth rate (k), but is nearly five‐ times more rapid, on average, in birds than in mammals.kGandkhave similar magnitude in birds, butkGis relatively much slower thankin mammals.

7. Rate of actuarial senescenceω(year−1), measured by the increase in mortality rate with age, is positively related to the rate of embryo growth in both birds and mammals, but is higher in the latter. Moreover, rate of ageing in birds is uniquely related to embryo growth while that in mammals is uniquely related to postnatal growth rate. Thus, development and ageing appear to be differently connected in birds and mammals, although the basis for these relationships is not known.
Early activation of invariant natural killer T cells in a rheumatoid arthritis model and application to disease treatment
Authors: Miellot‐Gafsou Aurore , Biton Jérôme , Bourgeois Elvire , Herbelin André , Boissier Marie‐Christophe , Bessis Natacha
Published: January 2010

Invariant NKT (iNKT) cells are a distinctive subtype of CD1d‐restricted T cells involved in regulating autoimmunity and capable of producing various T helper type 1 (Th1), Th2 and Th17 cytokines. Activation of iNKT cells by their exogenous ligand α‐galactosylceramide (α‐GalCer) exerts therapeutic effects in autoimmune diseases such as rheumatoid arthritis (RA). However, the pathophysiological role of iNKT cells in RA, in the absence of exogenous stimulation, is incompletely understood. We investigated the potential pathophysiological effects of iNKT cells in mice with collagen‐induced arthritis (CIA), a model of RA. We found that iNKT cells underwent activation only in the early phases of the disease (6 days post‐induction). In the liver, but not the spleen or lymph nodes, this early activation led to the release of interleukins ‐4, ‐17A and ‐10 and of interferon‐γ; and an increased CD69 expression. Importantly, clinical and histological signs of arthritis were improved by the functional blockade of iNKT cells by a monoclonal antibody to CD1d at the early phase of the disease. This improvement was associated on day 6 post‐induction with decreased expression of co‐stimulatory molecules (CD80, CD86, CD40) on splenic dendritic cells and macrophages, whereas regulatory T‐cell suppressive effects and proportions were not modified. Taken in concert, these findings suggest that iNKT cells are activated early in the course of CIA and contribute to the pathogenesis of arthritis. Therefore, iNKT‐cell activation may be a valid treatment target in RA.
Bowman‐Birk inhibitor affects pathways associated with energy metabolism in Drosophila melanogaster
Authors: Li H.‐M. , Sun L. , Mittapalli O. , Muir W. M. , Xie J. , Wu J. , Schemerhorn B. J. , Jannasch A. , Chen J. Y. , Zhang F. , Adamec J. , Murdock L. L. , Pittendrigh B. R.
Published: January 2010

Bowman‐Birk inhibitor (BBI) is toxic when fed to certain insects, including the fruit fly, Drosophila melanogaster. Dietary BBI has been demonstrated to slow growth and increase insect mortality by inhibiting the digestive enzymes trypsin and chymotrypsin, resulting in a reduced supply of amino acids. In mammals, BBI influences cellular energy metabolism. Therefore, we tested the hypothesis that dietary BBI affects energy‐associated pathways in the D. melanogaster midgut. Through microarray and metabolomic analyses, we show that dietary BBI affects energy utilization pathways in the midgut cells of D. melanogaster. In addition, ultrastructure studies indicate that microvilli are significantly shortened in BBI‐fed larvae. These data provide further insights into the complex cellular response of insects to dietary protease inhibitors.
A Rapid Interference between Glucocorticoids and cAMP‐Activated Signalling in Hypothalamic Neurones Prevents Binding of Phosphorylated cAMP Response Element Binding Protein and Glucocorticoid Receptor at the CRE‐Like and Composite GRE Sites of Thyrotrophin‐Releasing Hormone Gene Promoter
Authors: Díaz‐Gallardo M. Y. , Cote‐Vélez A. , Charli J. L. , Joseph‐Bravo P.
Published: January 2010

Glucocorticoids or cAMP increase, within minutes, thyrotrophin‐releasing hormone (TRH) transcription in hypothalamic primary cultures, although this effect is prevented if cells are simultaneously incubated with both drugs. Rat TRH promoter contains a CRE site at −101/−94 bp and a composite GRE element (cGRE) at −218/−197 bp. Nuclear extracts of hypothalamic cells incubated with 8Br‐cAMP or dexamethasone, and not their combination, bind to oligonucleotides containing the CRE or cGRE sequences. Adjacent to CRE are Sp/Krüppel response elements, and flanking the GRE half site, two AP1 binding sites. The present study aimed to identify the hypothalamic transcription factors that bind to these sites. We verified that the effects of glucocorticoid were not mimicked by corticosterone‐bovine serum albumin. Footprinting and chromatin immunoprecipitation (ChIP) assays were used to examine the interaction of cAMP‐ and glucocorticoid‐mediated regulation of TRH transcription at the CRE and cGRE regions of the TRH promoter. Nuclear extracts from hypothalamic cells incubated for 1 h with cAMP or glucocorticoids protected CRE. The GRE half site was recognised by nuclear proteins from cells stimulated with glucocorticoids and, for the adjacent AP‐1 sites, by nuclear proteins from cells stimulated with cAMP or phorbol esters. Protection of CRE or cGRE was lost if cells were coincubated with dexamethasone and 8Br‐cAMP. ChIP assays revealed phospho‐CREB, c‐Jun, Sp1, c‐Fos and GR antibodies bound the TRH promoter of cells treated with cAMP or glucocorticoids; anti:RNA‐polymerase II immunoprecipitated TRH promoter in a similar proportion as anti:pCREB or anti:GR. Recruitment of pCREB, SP1 or GR was lost when cells were exposed simultaneously to 8Br‐cAMP and glucocorticoids. The data show that while pCREB and Sp1 bind to CRE‐2, or GR to cGRE of the TRH promoter, the mutual antagonism between cAMP and glucocorticoid signalling, which prevent their binding to TRH promoter, could serve as a mechanism by which glucocorticoids rapidly suppress cAMP and noradrenaline‐stimulated TRH transcription.
Prenatal Social Stress in the Rat Programmes Neuroendocrine and Behavioural Responses to Stress in the Adult Offspring: Sex‐Specific Effects
Authors: Brunton P. J. , Russell J. A.
Published: January 2010

Stress exposure during pregnancy can ‘programme’ adult behaviour and hypothalamic‐pituitary‐adrenal (HPA) axis stress responsiveness. In the present study, we utilised an ethologically relevant social stressor to model the type of stress that pregnant women may experience. We investigated the effects of social defeat by a resident lactating rat over 5 days during the last week of pregnancy on the pregnant intruder rat HPA axis, and on HPA responsivity to stress and anxiety‐related behaviour in the adult offspring of the socially‐defeated intruder rats. HPA axis responses after social defeat were attenuated in the pregnant rats compared to virgin females. In the adult offspring, systemic interleukin (IL)‐1β or restraint increased adrenocorticotrophic hormone and corticosterone secretion in male and female control rats; however, in prenatally stressed (PNS) offspring, HPA responses were greatly enhanced and peak hormone responses to IL‐1β were greater in females versus males. Male PNS rats displayed increased anxiety behaviour on the elevated plus maze; however, despite marked changes in anxiety behaviour across the oestrous cycle, there were no differences between female control and PNS rats. Investigation of possible mechanisms showed mineralocorticoid mRNA levels were reduced in the hippocampus of male and female PNS offspring, whereas glucocorticoid receptor mRNA expression was modestly reduced in the CA2 hippocampal subfield in female PNS rats only. Corticotropin‐releasing hormone mRNA and glucocorticoid receptor mRNA expression in the central amygdala was greater in PNS males and females compared to controls. The data obtained in the present study indicate that prenatal social stress differentially programmes anxiety behaviour and HPA axis responses to stress in male and female offspring. Attenuated glucocorticoid feedback mechanisms in the limbic system may underlie HPA axis hyper‐reactivity to stress in PNS offspring.
Ligands of boron in Pisum sativum nodules are involved in regulation of oxygen concentration and rhizobial infection
Authors: REGUERA MARÍA , WIMMER MONIKA , BUSTOS PILAR , GOLDBACH HEINER E. , BOLAÑOS LUIS , BONILLA ILDEFONSO
Published: January 2010

Boron (B) is an essential nutrient for N2‐fixing legume–rhizobia symbioses, and the capacity of borate ions to bind and stabilize biomolecules is the basis of any B function. We used a borate‐binding‐specific resin and immunostaining techniques to identify B ligands important for the development of Pisum sativum–Rhizobium leguminosarum 3841 symbiotic nodules. arabinogalactan–extensin (AGPE), recognized by MAC 265 antibody, appeared heavily bound to the resin in extracts derived from B‐sufficient, but not from B‐deficient nodules. MAC 265 stained the infection threads and the extracellular matrix of cortical cells involved in the oxygen diffusion barrier. In B‐deprived nodules, immunolocalization of MAC 265 antigens was significantly reduced. Leghaemoglobin (Lb) concentration largely decreased in B‐deficient nodules. The absence of MAC 203 antigens in B‐deficient nodules suggests a high internal oxygen concentration, as this antibody detects an epitope on the lipopolysaccharide (LPS) of bacteroids typically expressed in micro‐aerobically grown R. leguminosarum 3841. However, B‐deprived nodules did not accumulate oxidized lipids and proteins, and revealed a decrease in the activity of the major antioxidant enzyme ascorbate peroxidase (APX). Therefore, B deficiency reduced the stability of nodule macromolecules important for rhizobial infection, and for regulation of oxygen concentration, resulting in non‐functional nodules, but did not appear to induce oxidative damage in low‐B nodules.
Group Structure, Nest Size and Reproductive Success in the Cooperatively Breeding Cichlid Julidochromis ornatus: A Correlation Study
Authors: Awata Satoshi , Kohda Masanori , Shibata Jun‐Ya , Hori Michio , Heg Dik
Published: January 2010

The effect of group size on reproductive success has long been studied in cooperatively breeding species, as it might provide an adaptive explanation for group‐living in social species. Numerous studies have shown positive effects of subordinates on reproductive success (‘helper effect’), but these studies have also revealed the importance of controlling statistically, or experimentally, for the effect of other factors that might affect reproductive success. Here, we first examine the relationships between group size, body size of group members and nest size in the cooperatively breeding cichlid Julidochromis ornatus, in which unrelated helpers frequently participate in reproduction and their breeding nests inside rock crevices may be crucial for reproduction and survival of all group members. Then, we subsequently investigate the relationship between group size and reproductive success, while controlling for these factors. The results showed that group size was significantly related to body size of group members rather than nest size; and larger breeders had larger helpers. It was found that group size significantly increased group reproductive output. More importantly, reproductive success of male breeders did not depend on the presence of mature helpers, whereas female reproductive success increased when two males assisted her and tended to decrease when two females bred cooperatively. We conclude that breeding groups of J. ornatus have size hierarchical societies that relate to group size, and group composition of genetically unrelated and co‐breeding members affects their reproductive success.
Development of sex‐linked AFLP markers in Simmondsia chinensis
Authors: Agarwal M. , Shrivastava N. , Padh H.
Published: January 2010

With 2 figures

In most economically important dioecious plants such as Simmondsia chinensis (Jojoba), sex identification based on flowering can only be made after 1–5 years of plantation. Identification of sex‐associated molecular markers for ascertaining sex at seedling stage is a desirable prerequisite for optimal plantation and economical benefits. Jojoba is being developed as a plantation crop in the arid regions of Western India due to the commercial benefits of the unique waxy content of its seeds. In the present study, sex‐linked DNA markers have been identified in S. chinensis using the AFLP technique. From sixteen primers combinations used for AFLP analysis, two combinations EcoRI‐TAC/MseI‐GCG and EcoRI‐GC/MseI‐GCG resulted in amplification of sex‐linked markers. Two male‐specific markers of ∼525 bp and ∼325 bp were identified using the primer combinations EcoRI‐GC/MseI‐GCG and EcoRI‐TAC/MseI‐GCG, respectively. A female‐specific marker of ∼270 bp was also identified with the primer combination EcoRI‐TAC/MseI‐GCG. In future, SCAR markers developed from these sex‐specific markers will provide an effective and reliable method for sex identification.
Characteristics and EGFP Expression of Goat Mammary Gland Epithelial Cells
Authors: Zheng Y‐M , He X‐Y , Zhang Y
Published: January 2010

Contents

The aims of this study were (i) to establish a goat mammary gland epithelial (GMGE) cell line, and (ii) to determine if these GMGE cells could be maintained long‐term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of GMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating goat. The passage 16 GMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in GMGE cells was test by immunofluorescence. Βeta‐Casein gene mRNA was test for GMGE cells by RT‐PCR. The results showed that when grown at low density on a plastic substratum, the GMGE cells formed islands, and when grown to confluency, the cells formed a monolayer and aggregated with the characteristic cobble‐stone morphology of epithelial cells. GMGE cells could form dome‐like structure which looked like nipple, and the lumen‐like structures formed among the cells. Several blister‐like structures appeared in the appearance of the cells. The GMGE cells contained different cell types, majority of the cells were short shuttle‐like or polygon which were beehive‐like. A part of cells were round and flat, a small number of cells were elongated. Some of the GMGE cells contained milk drops. The cell nuclei were round which had 2–4 obvious cores. The expression of Cell keratins demonstrated the property of epithelial cells in GMGE cells by immunofluorescence. The GMGE cells could express transcript encoding a Βeta‐Casein protein. EGFP gene was successfully transferred into the GMGE cells, and the transfected cells could be maintained long‐term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected GMGE (ET‐GMGE) cell line and maintained it long‐term in culture by continuous subculturing.
The Cryoprotective Effects of Vitamin B12 Supplementation on Bovine Semen Quality
Authors: Hu J‐H , Tian W‐Q , Zhao X‐L , Zan L‐S , Xin Y‐P , Li Q‐W
Published: January 2010

Contents

The present study aimed to investigate the effects of vitamin B12 supplementation on standard bovine semen quality parameters and anti‐oxidative enzyme activities. Vitamin B12 was supplemented at concentrations of 1.25, 2.5, 3.75 and 5.0 mg/ml to bovine semen cryoprotective medium. The results indicated that the motility and straight line velocity, curvilinear velocity, mean coefficient, velocity of the average path values of sperm supplemented with 2.50 mg/ml vitamin B12 were significantly higher than that of other groups (p < 0.05). No significant difference was observed for linearity index, lateral head displacement values and the percentage of grade A spermatozoa between the extenders containing 2.50 and 3.75 mg/ml vitamin B12 (p > 0.05). The percentages of acrosome‐intact and plasma membrane‐intact spermatozoa were significantly improved (p < 0.05) by supplementing with 2.50 mg/ml vitamin B12. The results of biochemical assay revealed that vitamin B12 supplementation did not cause significant changes in superoxide dismutase levels compared with control (p > 0.05). However, the catalase levels were higher in the treatment supplemented with vitamin B12 at 2.50 mg/ml, when compared with other groups (p < 0.05). The extender supplemented with vitamin B12 significantly decreased glutathione peroxidase activity compared with the control (p < 0.05). The supplementation of 3.75 mg/ml vitamin B12 caused the highest value of glutathione reductase activity, compared with other groups (p < 0.05). In conclusion, the extender supplemented with vitamin B12 could reduce the oxidative stress provoked by freezing‐thawing and improve bovine semen quality. Further studies are required to obtain more concrete results on the determination of lipid peroxidation and antioxidant capacities of vitamin B12 in cryopreserved bovine semen.
Environmental risk factors in inflammatory bowel disease
Authors: Leong Rupert W L
Published: January 2010

See article in J. Gastroenterol. Hepatol. 2010; 25: 325–333