bluuebunny/crossref_metadata_2025 huggingface dataset:
Both Saturated and n-6 Polyunsaturated Fat Diets Reduce Phosphorylation of Insulin Receptor Substrate-1 and Protein Kinase B in Muscle during the Initial Stages of in Vivo Insulin Stimulation
Authors: Frangioudakis Georgia , Ye Ji-Ming , Cooney Gregory J.
Published: September 2005

Our aim was to determine the importance of changes in phosphorylation of key insulin signaling intermediates in the insulin resistance observed in skeletal muscle of rats fed diets high in saturated or n-6 polyunsaturated fat. We used phospho-specific antibodies to measure the time course of phosphorylation of key components of the insulin signaling pathway by immunoblotting during the initial stages of a physiological elevation in the circulating insulin concentration. The phosphorylation of insulin receptor at Tyr1162/1163 (IR Tyr1162/1163) increased over 20 min of insulin infusion, whereas the downstream phosphorylation of insulin receptor substrate-1 Tyr612 (IRS-1 Tyr612) peaked at 5 min and declined thereafter. Interestingly, phosphorylation of IRS-1 at Tyr895 continued to increase over the 20-min period, and protein kinase B (PKB) phosphorylation at Ser473 reached a plateau by 5 min, demonstrating that different profiles of phosphorylation are involved in transmission of the insulin signal despite a constant level of insulin stimulation. In muscle from rats fed high n-6 polyunsaturated or saturated fat diets, however, there was no insulin-stimulated increase in IRS-1 Tyr612 phosphorylation and a temporal difference in PKB Ser473 phosphorylation despite no difference in IR Tyr1162/1163 phosphorylation, IRS-1 Tyr895 phosphorylation, and ERK phosphorylation. These results demonstrate that under conditions of increased insulin, similar to those used to assess insulin action in vivo, chronic high-fat feeding impairs insulin signal transduction related to glucose metabolism at the level of IRS-1 Tyr612 and PKB Ser473 and that these effects are independent of the type of fat used in the high-fat diet.
Differential Biosynthesis and Intracellular Transport of Follistatin Isoforms and Follistatin-Like-3
Authors: Saito Seiichiro , Sidis Yisrael , Mukherjee Abir , Xia Yin , Schneyer Alan
Published: September 2005

Follistatin (FST) and FST-like-3 (FSTL3) are structurally related proteins that bind and neutralize activin and closely related members of the TGFβ superfamily. Three FST isoforms (FST288, FST303, and FST315) are produced from the Fst gene that are primarily secreted proteins. FSTL3 is secreted, but is also observed within the nucleus of most cells. We used pulse-chase 35S labeling to examine the biosynthetic and intracellular transport patterns that lead to differential secretion and intracellular retention of these proteins. Among the FST isoforms, FST315 was secreted fastest and FST288 was secreted more slowly, with some remaining intracellular. In contrast, FSTL3 was secreted the slowest, with newly synthesized proteins being both secreted and trafficked to the nucleus. This nuclear FSTL3 was N-glycosylated, although not to the same degree as secreted FSTL3. Both FST and FSTL3 have two Mets in their signal sequence. Mutation of the first Met in FST288 eliminated protein translation, whereas FSTL3 could be translated from either Met. However, although FSTL3 translated from the second Met, which had no signal sequence, was confined to the nucleus, it was not glycosylated. Interestingly, this FSTL3 retained activin-antagonizing activity. Thus, although bioactive, nuclear FSTL3 can be translated from the second Met when the first Met is mutated, the glycosylated nuclear FSTL3 produced endogenously indicates that a different mechanism must be used under natural conditions that apparently includes N-glycosylation. Moreover, the differential biosynthetic and intracellular transport patterns for FST288 and FSTL3 suggest that these two activin-binding proteins may have distinct intracellular roles.
Dynamics of in Vivo Release of Molt-Inhibiting Hormone and Crustacean Hyperglycemic Hormone in the Shore Crab, Carcinus maenas
Authors: Chung J. S. , Webster S. G.
Published: September 2005

Very little is known regarding the release patterns or circulating titers of neuropeptides in crustaceans, in particular those concerned with regulation of molting hormone (ecdysteroid) synthesis, molt-inhibiting hormone (MIH), and crustacean hyperglycemic hormone (CHH), which is also an adaptive hormone, centrally important in carbohydrate metabolism. Furthermore, the currently accepted model of molt control is founded on an untested hypothesis suggesting that molting can proceed only after decline in MIH titer. Accordingly, we measured simultaneous circulating neuropeptide profiles for both MIH and CHH by RIA of purified hemolymph during the molt cycle at fine temporal scale during day/night cycles and seasonally. For CHH we additionally determined release patterns after physiologically relevant stress. Results show that both hormones are released exclusively and episodically, rather than continuously, with notably short half-lives in circulation, suggesting dynamic and short-lived variations in levels of both hormones. During the molt cycle, there are no overt changes in MIH titer, except a massive and unprecedented increase in MIH during late premolt, just before ecdysis. The function of this hormone surge is unknown. Treatment with various stressors (hypoxia, temperature shock) showed that CHH release occurs extremely rapidly, within minutes of stress. Release of CHH after stressful episodes during premolt (when gut endocrine cells synthesize large quantities of CHH) is exclusively from the sinus gland: CHH from the gut is never involved in the stress response. The results show a hitherto unsuspected dynamism in release of MIH and CHH and suggest that currently accepted models of molt control must be reconsidered.
Tissue Deiodinase Activity during Prolonged Critical Illness: Effects of Exogenous Thyrotropin-Releasing Hormone and Its Combination with Growth Hormone-Releasing Peptide-2
Authors: Debaveye Yves , Ellger Björn , Mebis Liese , Van Herck Erik , Coopmans Willy , Darras Veerle , Van den Berghe Greet
Published: September 2005

Prolonged critical illness is characterized by reduced pulsatile TSH secretion, causing reduced thyroid hormone release and profound changes in thyroid hormone metabolism, resulting in low circulating T3 and elevated rT3 levels. To further unravel the underlying mechanisms, we investigated the effects of exogenous TRH and GH-releasing peptide-2 (GHRP-2) in an in vivo model of prolonged critical illness. Burn-injured, parenterally fed rabbits were randomized to receive 4-d treatment with saline, 60 μg/kg·h GHRP-2, 60 μg/kg·h TRH, or 60 μg/kg·h TRH plus 60 μg/kg·h GHRP-2 started on d 4 of the illness (n = 8/group). The activities of the deiodinase 1 (D1), D2, and D3 in snap-frozen liver, kidney, and muscle as well as their impact on circulating thyroid hormone levels were studied. Compared with healthy controls, hepatic D1 activity in the saline-treated, ill animals was significantly down-regulated (P = 0.02), and D3 activity tended to be up-regulated (P = 0.06). Infusion of TRH and TRH plus GHRP-2 restored the catalytic activity of D1 (P = 0.02) and increased T3 levels back within physiological range (P = 0.008). D3 activity was normalized by all three interventions, but only addition of GHRP-2 to TRH prevented the rise in rT3 seen with TRH alone (P = 0.02). Liver D1 and D3 activity were correlated (respectively, positively and negatively) with the changes in circulating T3 (r = 0.84 and r = −0.65) and the T3/rT3 ratio (r = 0.71 and r = −0.60). We conclude that D1 activity during critical illness is suppressed and related to the alterations within the thyrotropic axis, whereas D3 activity tends to be increased and under the joint control of the somatotropic and thyrotropic axes.
Loss of Synaptonemal Complex Protein-1, a Synaptonemal Complex Protein, Contributes to the Initiation of Follicular Assembly in the Developing Rat Ovary
Authors: Paredes Alfonso , Garcia-Rudaz Cecilia , Kerr Bredford , Tapia Veronica , Dissen Gregory A. , Costa Maria E. , Cornea Anda , Ojeda Sergio R.
Published: September 2005

In the rat ovary, germ and somatic cells become organized into primordial follicles 48–72 h after birth. Although several genes have been implicated in the control of early follicular growth, less is known about the factors involved in the formation of primordial follicles. Using the method of differential display of mRNAs, we found several genes differentially expressed at the time of follicular assembly. One of them encodes synaptonemal complex protein-1 (SCP1), a core component of the protein complex that maintains recombining chromosomes together during prophase I of the first meiotic division in germ cells. This association, evident during the pachytene stage, ends when chromosomal desynapsis begins in the diplotene stage at the end of prophase I. Oocytes become arrested in the diplotene/dictate stage before becoming enclosed into primordial follicles, suggesting that oocytes must complete meiotic prophase I before becoming competent to direct follicle assembly. We now show that attainment of the diplotene stage results in follicular formation. In developing rat ovaries, SCP1 mRNA expression is confined to oocytes and decreases precipitously within 24 h after birth, preceding the organization of primordial follicles. The premature loss of SCP1, achieved via treatment with an antisense oligodeoxynucleotide targeting SCP1 mRNA, resulted in more oocytes reaching the diplotene stage, as evidenced by a decrease in the number of oocytes containing germ cell nuclear antigen-1 (a nuclear protein whose expression ceases in diplotene) and an increase in the number of oocytes expressing MSY2 (a cytoplasmic Y box protein expressed in oocytes that have become arrested in diplotene). SCP1-deficient ovaries exhibited an increased number of newly formed follicles, suggesting that completion of meiotic prophase I endows oocytes with the ability to orchestrate follicular assembly.
Interleukin-1α Inhibits Insulin Signaling with Phosphorylating Insulin Receptor Substrate-1 on Serine Residues in 3T3-L1 Adipocytes
Authors: He Jianying , Usui Isao , Ishizuka Ken , Kanatani Yukiko , Hiratani Kazuyuki , Iwata Minoru , Bukhari Agussalim , Haruta Tetsuro , Sasaoka Toshiyasu , Kobayashi Masashi
Published: September 2005

Proinflammatory cytokines are recently reported to inhibit insulin signaling causing insulin resistance. IL-1α is also one of the proinflammatory cytokines; however, it has not been clarified whether IL-1α may also cause insulin resistance. Here, we investigated the effects of IL-1α treatment on insulin signaling in 3T3-L1 adipocytes. IL-1α treatment up to 4 h did not alter insulin-stimulated insulin receptor tyrosine phosphorylation, whereas tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and the association with phosphatidylinositol 3-kinase were partially inhibited with the maximal inhibition in around 15 min. IRS-1 was transiently phosphorylated on some serine residues around 15 min after IL-1α stimulation, when several serine kinases, IκB kinase, c-Jun-N-terminal kinase, ERK, and p70S6K were activated. Chemical inhibitors for these kinases inhibited IL-1α-induced serine phosphorylation of IRS-1. Tyrosine phosphorylation of IRS-1 was recovered only by the IKK inhibitor or JNK inhibitor, suggesting specific involvement of these two kinases. Insulin-stimulated Akt phosphorylation and 2-deoxyglucose uptake were not inhibited only by IL-1α. Interestingly, Akt phosphorylation was synergistically inhibited by IL-1α in the presence of IL-6. Taken together, short-term IL-1α treatment transiently causes insulin resistance at IRS-1 level with its serine phosphorylation. IL-1α may suppress insulin signaling downstream of IRS-1 in the presence of other cytokines, such as IL-6.
A Gja1 missense mutation in a mouse model of oculodentodigital dysplasia
Authors: Flenniken Ann M. , Osborne Lucy R. , Anderson Nicole , Ciliberti Nadia , Fleming Craig , Gittens Joanne E. I. , Gong Xiang-Qun , Kelsey Lois B. , Lounsbury Crystal , Moreno Luisa , Nieman Brian J. , Peterson Katie , Qu Dawei , Roscoe Wendi , Shao Qing , Tong Dan , Veitch Gregory I. L. , Voronina Irina , Vukobradovic Igor , Wood Geoffrey A. , Zhu Yonghong , Zirngibl Ralph A. , Aubin Jane E. , Bai Donglin , Bruneau Benoit G. , Grynpas Marc , Henderson Janet E. , Henkelman R. Mark , McKerlie Colin , Sled John G. , Stanford William L. , Laird Dale W. , Kidder Gerald M. , Adamson S. Lee , Rossant Janet
Published: September 2005

Oculodentodigital dysplasia (ODDD) is an autosomal dominant disorder characterized by pleiotropic developmental anomalies of the limbs, teeth, face and eyes that was shown recently to be caused by mutations in the gap junction protein alpha 1 gene (GJA1), encoding connexin 43 (Cx43). In the course of performing an N-ethyl-N-nitrosourea mutagenesis screen, we identified a dominant mouse mutation that exhibits many classic symptoms of ODDD, including syndactyly, enamel hypoplasia, craniofacial anomalies and cardiac dysfunction. Positional cloning revealed that these mice carry a point mutation in Gja1 leading to the substitution of a highly conserved amino acid (G60S) in Cx43. In vivo and in vitro studies revealed that the mutant Cx43 protein acts in a dominant-negative fashion to disrupt gap junction assembly and function. In addition to the classic features of ODDD,these mutant mice also showed decreased bone mass and mechanical strength, as well as altered hematopoietic stem cell and progenitor populations. Thus,these mice represent an experimental model with which to explore the clinical manifestations of ODDD and to evaluate potential intervention strategies.
Basolateral localization of native ClC-2 chloride channels in absorptive intestinal epithelial cells and basolateral sorting encoded by a CBS-2 domain di-leucine motif
Authors: Peña-Münzenmayer Gaspar , Catalán Marcelo , Cornejo Isabel , Figueroa Carlos D. , Melvin James E. , Niemeyer María I. , Cid L. Pablo , Sepúlveda Francisco V.
Published: September 2005

The Cl– channel ClC-2 is expressed in transporting epithelia and has been proposed as an alternative route for Cl– efflux that might compensate for the malfunction of CFTR in cystic fibrosis. There is controversy concerning the cellular and membrane location of ClC-2, particularly in intestinal tissue. The aim of this paper is to resolve this controversy by immunolocalization studies using tissues from ClC-2 knockout animals as control, ascertaining the sorting of ClC-2 in model epithelial cells and exploring the possible molecular signals involved in ClC-2 targeting. ClC-2 was exclusively localized at the basolateral membranes of surface colonic cells or villus duodenal enterocytes. ClC-2 was sorted to the basolateral membranes in MDCK, Caco-2 and LLC-PK1-μ1B, but not in LLC-PK1-μ1A cells. Mutating a di-leucine motif (L812L813) to a di-alanine changed the basolateral targeting of ClC-2 to an apical location. The basolateral membrane localization of ClC-2 in absorptive cells of the duodenum and the colon is compatible with an absorptive function for this Cl– channel. Basolateral targeting information is contained in a di-leucine motif (L812L813) within CBS-2 domain at the C-terminus of ClC-2. It is speculated that ClC-2 also contains an apical sorting signal masked by L812L813. The proposal that CBS domains in ClC channels might behave as regulatory sites sensing intracellular signals opens an opportunity for pharmacological modulation of ClC-2 targeting.
Measuring cell adhesion forces of primary gastrulating cells from zebrafish using atomic force microscopy
Authors: Puech Pierre-Henri , Taubenberger Anna , Ulrich Florian , Krieg Michael , Muller Daniel J. , Heisenberg Carl-Philipp
Published: September 2005

During vertebrate gastrulation, progenitor cells of different germ layers acquire specific adhesive properties that contribute to germ layer formation and separation. Wnt signals have been suggested to function in this process by modulating the different levels of adhesion between the germ layers, however, direct evidence for this is still lacking. Here we show that Wnt11, a key signal regulating gastrulation movements, is needed for the adhesion of zebrafish mesendodermal progenitor cells to fibronectin, an abundant extracellular matrix component during gastrulation. To measure this effect, we developed an assay to quantify the adhesion of single zebrafish primary mesendodermal progenitors using atomic-force microscopy (AFM). We observed significant differences in detachment force and work between cultured mesendodermal progenitors from wild-type embryos and from slb/wnt11 mutant embryos, which carry a loss-of-function mutation in the wnt11 gene, when tested on fibronectin-coated substrates. These differences were probably due to reduced adhesion to the fibronectin substrate as neither the overall cell morphology nor the cell elasticity grossly differed between wild-type and mutant cells. Furthermore, in the presence of inhibitors of fibronectin-integrin binding, such as RGD peptides, the adhesion force and work were strongly decreased, indicating that integrins are involved in the binding of mesendodermal progenitors in our assay. These findings demonstrate that AFM can be used to quantitatively determine the substrate-adhesion of cultured primary gastrulating cells and provide insight into the role of Wnt11 signalling in modulating cell adhesion at the single cell scale.
Depletion of GAK/auxilin 2 inhibits receptor-mediated endocytosis and recruitment of both clathrin and clathrin adaptors
Authors: Lee Dong-won , Zhao Xiaohong , Zhang Fang , Eisenberg Evan , Greene Lois E.
Published: September 2005

Cyclin G-associated kinase (GAK/auxilin 2), the ubiquitous form of the neuronal-specific protein auxilin 1, is an essential cofactor for the Hsc70-dependent uncoating of clathrin-coated vesicles. We have now investigated the effect of knocking down GAK in HeLa cells by vector-based small hairpin RNA. Functionally, depletion of GAK caused a marked decrease in internalization of both transferrin and epidermal growth factor and altered mannose 6-phosphate receptor trafficking, but had little effect on the recycling of transferrin receptor back to the plasma membrane. Structurally, depletion of GAK caused a marked reduction in perinuclear clathrin associated with the trans-Golgi network and in the number of clathrin-coated pits on the plasma membrane, and reduced clathrin exchange on the few clathrin-coated pits that remained. Surprisingly, while clathrin depletion does not prevent adaptors from assembling on the membrane, depletion of GAK caused a dramatic reduction in AP2 and epsin on the plasma membrane and AP1 and GGA at the trans-Golgi network. A similar effect was caused by expression of a dominant negative Hsp70 mutant. These results suggest that GAK, in conjunction with Hsc70, not only uncoats clathrin-coated vesicles and induces clathrin exchange on clathrin-coated pits, but also mediates binding of clathrin and adaptors to the plasma membrane and the trans-Golgi network.
Paxillin is required for cell-substrate adhesion, cell sorting and slug migration duringDictyosteliumdevelopment
Authors: Bukahrova Tanya , Weijer Gertrud , Bosgraaf Leonard , Dormann Dirk , van Haastert Peter J. , Weijer Cornelis J.
Published: September 2005

Paxillin is a key regulatory component of focal adhesion sites, implicated in controlling cell-substrate interactions and cell movement. We analyse the function of aDictyostelium discoideumpaxillin homologue, PaxB, which contains four highly conserved LD and four LIM domains, but lacks two characteristic tyrosine residues, that form the core of vertebrate SH2-binding domains. PaxB is expressed during growth and all stages of development, but expression peaks during slug formation. Using apaxB-gfpknockin strain we show the existence of focal adhesions and characterise their dynamics. During multicellular development PaxB is not only found in focal adhesions at the cell-substrate interface, but also in the tips of filopodial structures predominantly located at the trailing ends of cells.paxB–strains are less adhesive to the substrate, they can aggregate but multicellular development from the mound stage onwards is severely impeded.paxB– strains are defective in proper cell type proportioning, cell sorting, slug migration and form-defective fruiting bodies. Mutation of a conserved JNK phosphorylation site, implicated in the control of cell migration, does not have any major effects on cell sorting, slug migration or morphogenesis inDictyostelium. PaxB does not appear to function redundantly with its closest relative Lim2 (paxA), which when deleted also results in a mound arrest phenotype. However, analysis ofpaxA–andpaxB–single and double null mutants suggest that PaxB may act upstream of Lim2.
Variable rewards and discrimination ability in an insect herbivore: what and how does a hungry locust learn?
Authors: Behmer Spencer T. , Belt Corlisa E. , Shapiro Martin S.
Published: September 2005

With the exception of honeybees, there have been few good invertebrate models for associative learning. Grasshoppers and locusts (Orthoptera:Acrididae) possess a number of characteristics that make them excellent candidates for such studies, and in this paper we present a novel protocol,based on a Y-maze, that is specifically designed for studying their learning and choice behaviour. Three separate experiments were conducted using individual gregarious forms of the desert locust, Schistocerca gregaria. In our first experiment, coloured arms of a two-sided Y-maze provided a large or small amount of wheat for nine choice-trials. In the second experiment, locusts discriminated odours with wheat rewards for nine choice-trials. The odour-wheat reward combinations were then reversed for an additional nine choice-trials. For the third experiment, the locusts again discriminated odours, but here we used artificial foods and the rewards differed in their concentration of protein and digestible carbohydrate. The results indicate that, in addition to showing good acquisition of choice performance, the locusts also took less time to reach the larger-rewarded option. The data indicate that our protocol is highly sensitive for recording choice behaviour in acridids and reveals the potential they have for advancing our current understanding of associative learning and the field of learning in general.
Female receptivity in butterflies and moths
Authors: Wedell Nina
Published: September 2005

Female receptivity in butterflies and moths is influenced by a multitude of factors that vary between virgin and mated females, and is often affected by the quality and persistence of courting males. Mated females of polyandrous species frequently display a period of non-receptivity following mating, often resulting from factors transferred by the male at mating. Some of these compounds have a transient effect (e.g. anti-aphrodisiacs and mating plugs),whereas others induce long-term suppression of receptivity (i.e. sperm and seminal factors). Sperm appear to generally induce long-term suppression of female receptivity in both butterflies and moths. In some species, production of non-fertile sperm may function to fill the female's sperm storage organ and switch off receptivity, although whether this is a general phenomenon across the Lepidoptera has not yet been examined. Examination of seminal fluids suppressing female receptivity in moths suggests that more than one factor is implicated, but frequently the transfer or stimulation of Juvenile Hormone production is involved. Surprisingly, potential seminal factors influencing female receptivity in butterflies remain largely unexplored. In this review, I summarize the various factors that are known to affect female receptivity in the Lepidoptera to date, and briefly compare the function and similarity of the Pheromone Suppressing Peptide (HezPSP) in moths to that of the Sex Peptide in Drosophila melanogaster (DrmSP). The exciting possibility that seminal peptides in the Lepidoptera and Diptera (e.g. Drosophila melanogaster) may have shared functionality is discussed.
Energy metabolism in orchid bee flight muscles: carbohydrate fuels all
Authors: Suarez Raul K. , Darveau Charles-A. , Welch Kenneth C. , O'Brien Diane M. , Roubik David W. , Hochachka Peter W.
Published: September 2005

The widely accepted idea that bees fuel flight through the oxidation of carbohydrate is based on studies of only a few species. We tested this hypothesis as part of our research program to investigate the size-dependence of flight energetics in Panamanian orchid bees. We succeeded in measuring rates of O2 consumption and CO2 production in vivo during hovering flight, as well as maximal activities(Vmax values) in vitro of key enzymes in flight muscle energy metabolism in nine species belonging to four genera. Respiratory quotients (ratios of rates of CO2 production to O2consumption) in all nine species are close to 1.0. This indicates that carbohydrate is the main fuel used for flight. Trehalase, glycogen phosphorylase and hexokinase activities are sufficient to account for the glycolytic flux rates estimated from rates of CO2 production. High activities of other glycolytic enzymes, as well as high activities of mitochondrial oxidative enzymes, are consistent with the estimated rates of carbohydrate-fueled oxidative metabolism. In contrast, hydroxyacylCoA dehydrogenase, an enzyme involved in fatty acid oxidation, was not detectable in any species. Thoracic homogenates displayed ADP-stimulated oxidition of pyruvate + proline, but did not oxidize palmitoyl l-carnitine +proline as substrates. A metabolic map, based on data reported herein and information from the literature, is presented. The evidence available supports the hypothesis that carbohydrate serves as the main fuel for flight in bees.
Allometric scaling of flight energetics in orchid bees: evolution of flux capacities and flux rates
Authors: Darveau Charles-A. , Hochachka Peter W. , Roubik David W. , Suarez Raul K.
Published: September 2005

The evolution of metabolic pathways involved in energy production was studied in the flight muscles of 28 species of orchid bees. Previous work revealed that wingbeat frequencies and mass-specific metabolic rates decline in parallel by threefold as body mass increases interspecifically over a 20-fold range. We investigated the correlated evolution of metabolic rates during hovering flight and the flux capacities, i.e. Vmaxvalues, of flight muscle enzymes involved in substrate catabolism, the Krebs cycle and the electron transport chain. Vmax at the hexokinase (HK) step scales allometrically with an exponent almost identical to those obtained for wingbeat frequency and mass-specific metabolic rate. Analysis of this relationship using phylogenetically independent contrasts supports the hypothesis of correlated evolution between HK activity and mass-specific metabolic rate. Although other enzymes scale allometrically with respect to body mass, e.g. trehalase, glycogen phosphorylase and citrate synthase, no other enzyme activities were correlated with metabolic rate after controlling for phylogenetic relatedness. Pathway flux rates were used with enzyme Vmax values to estimate fractional velocities(fraction of Vmax at which enzymes operate) for various reactions to gain insights into enzyme function and how this varies with body mass. Fractional velocity is highly conserved across species at the HK step,but varied at all other steps examined. These results are discussed in the context of the regulation and evolution of pathways of energy metabolism.
Roles of hierarchical and metabolic regulation in the allometric scaling of metabolism in Panamanian orchid bees
Authors: Suarez Raul K. , Darveau Charles-A. , Hochachka Peter W.
Published: September 2005

Assessment of the relative importance of variation in enzyme concentration[E] and metabolic regulation in accounting for interspecific variation in metabolic rates is an unrealized area of research. Towards this end, we used metabolic flux rates during hovering and enzymatic flux capacities(Vmax values, equal to [E]×kcat,where kcat is catalytic efficiency) in flight muscles measured in vitro from 14 orchid bee species ranging in body mass from 47 to 1065 mg. Previous studies revealed that, across orchid bee species,wingbeat frequencies and metabolic rates decline in parallel with increasing body mass. Vmax values at some enzymatic steps in pathways of energy metabolism decline with increasing mass while, at most other steps, Vmax values are mass-independent. We quantified the relative importance of `hierarchical regulation' (alteration in Vmax, indicative of alteration in [E]) and `metabolic regulation' (resulting from variation in substrate, product or modulator concentrations) in accounting for interspecific variation in flux across species. In addition, we applied the method of phylogenetically independent contrasts to remove the potentially confounding effects of phylogenetic relationships among species. In the evolution of orchid bees, hierarchical regulation completely accounts for allometric variation in flux rates at the hexokinase step while, at other reactions, variation in flux is completely accounted for by metabolic regulation. The predominant role played by metabolic regulation is examined at the phosphoglucoisomerase step using the Haldane relationship. We find that extremely small variation in the concentration ratio of [product]/[substrate] is enough to cause the observed interspecific variation in net flux at this reaction in glycolysis.
Energetic costs of diving and thermal status in European shags(Phalacrocorax aristotelis)
Authors: Enstipp Manfred R. , Grémillet David , Lorentsen Svein-Håkon
Published: September 2005

Diving is believed to be very costly in cormorants (Phalacrocoracidae) when compared with other avian divers because of their poor insulation and less-efficient foot propulsion. It was therefore suggested that cormorants might employ a behavioural strategy to reduce daily energy expenditure by minimizing the amount of time spent in water. However, European shags(Phalacrocorax aristotelis) have been observed to spend up to 7 h day-1 diving in water of around 5-6°C. To gain a better understanding of the energetic requirements in European shags, we measured their metabolic rates when resting in air/water and during shallow diving using respirometry. To investigate the effects of water temperature and feeding status on metabolic rate, birds dived at water temperatures ranging from 5 to 13°C in both post-absorptive and absorptive states. In parallel with respirometry, stomach temperature loggers were deployed to monitor body temperature. Basal metabolic rate (BMR) was almost identical to allometric predictions at 4.73 W kg-1. Metabolic rate when resting on water, during diving and after feeding was significantly elevated when compared with the resting-in-air rate. During diving, the metabolic rate of post-absorptive shags increased to 22.66 W kg-1, which corresponds to 4.8× BMR. Minimum cost of transport (COT) was calculated at 17.8 J kg-1 m-1 at a swim speed of 1.3 m s-1. Feeding before diving elevated diving metabolic rate by 13%for up to 5 h. There was a significant relationship between diving metabolic rate and water temperature, where metabolic rate increased as water temperature declined. Thermal conductance when resting in air at 10-19°C was 2.05 W m-2 °C-1 and quadrupled during diving(7.88 W m-2 °C-1). Stomach temperature when resting in air during the day was 40.6°C and increased during activity. In dive trials lasting up to 50 min, stomach temperature fluctuated around a peak value of 42.0°C. Hence, there is no evidence that European shags might employ a strategy of regional hypothermia. The energetic costs during shallow diving in European shags are considerably lower than has previously been reported for great cormorants (Phalacrocorax carbo) and are comparable to other foot-propelled divers. The lower dive costs in shags might be the consequence of a more streamlined body shape reducing hydrodynamic costs as well as a greater insulative plumage air layer (estimated to be 2.71 mm), which reduces thermoregulatory costs. The latter might be of great importance for shags especially during winter when they spend extended periods foraging in cold water.
Environmental hypoxia influences hemoglobin subunit composition in the branchiopod crustaceanTriops longicaudatus
Authors: Guadagnoli J. A. , Braun A. M. , Roberts S. P. , Reiber C. L.
Published: September 2005

Hemoglobin (Hb) is a highly conserved protein that provides a vital link between environmental oxygen and its use and/or storage within an organism. While ubiquitous among vertebrates, Hb occurs frequently in invertebrate phyla as well. Many arthropod species use the copper-binding pigment hemocyanin, but unique in this phylum are the branchiopod crustaceans, which express Hb. Branchiopod Hb concentration and structure are exquisitely sensitive to environmental oxygen availability. Hemoglobin concentration and oxygen-binding affinity increase with decreasing oxygen tension in Daphnia, Artemia and Triops. The change in binding affinity is attributed to differential Hb subunit expression in Daphnia and Artemia but remains unclear for Triops. This is the first study to demonstrate developmental plasticity of Hb subunit expression in a notostracan, Triops longicaudatus, reared under conditions of varying oxygen availability. In response to variable oxygen environments, T. longicaudatus differentially express four primary Hb subunits ranging between 30 and 34 kDa, with normoxic-reared animals expressing primarily the heavier subunits, and hypoxic-reared animals expressing increased proportions of the lower molecular mass subunits. Moreover, differential Hb subunit expression is induced upon transfer of normoxic-reared adults to a hypoxic environment, such that the distribution of Hb subunits in the transferred adults becomes similar to that of hypoxic-reared animals. Two-dimensional gel electrophoresis and follow-up analyses revealed several isoforms of Hb subunits that may represent differential gene expression and/or post-translational modification. Unlike Daphnia and Artemia,the Hb hypoxic response in Triops is not reversible in that there was no significant decrease in Hb concentration or change in Hb subunit expression pattern when hypoxic-reared adults were transferred to a normoxic environment.
Olfactory coding in Drosophila larvae investigated by cross-adaptation
Authors: Boyle Jennefer , Cobb Matthew
Published: September 2005

In order to reveal aspects of olfactory coding, the effects of sensory adaptation on the olfactory responses of first-instar Drosophila melanogaster larvae were tested. Larvae were pre-stimulated with a homologous series of acetic esters (C3-C9), and their responses to each of these odours were then measured. The overall patterns suggested that methyl acetate has no specific pathway but was detected by all the sensory pathways studied here, that butyl and pentyl acetate tended to have similar effects to each other and that hexyl acetate was processed separately from the other odours. In a number of cases, cross-adaptation transformed a control attractive response into a repulsive response; in no case was an increase in attractiveness observed. This was investigated by studying changes in dose-response curves following pre-stimulation. These findings are discussed in light of the possible intra- and intercellular mechanisms of adaptation and the advantage of altered sensitivity for the larva.
Biological Variation of Vitamins in Blood of Healthy Individuals
Authors: Talwar Dinesh K , Azharuddin Mohammed K , Williamson Cathy , Teoh Yee Ping , McMillan Donald C , St. J. O’Reilly Denis
Published: September 2005

Background: Components of biological variation can be used to define objective quality specifications (imprecision, bias, and total error), to assess the usefulness of reference values [index of individuality (II)], and to evaluate significance of changes in serial results from an individual [reference change value (RCV)]. However, biological variation data on vitamins in blood are limited. The aims of the present study were to determine the intra- and interindividual biological variation of vitamins A, E, B1, B2, B6, C, and K and carotenoids in plasma, whole blood, or erythrocytes from apparently healthy persons and to define quality specifications for vitamin measurements based on their biology.

Methods: Fasting plasma, whole blood, and erythrocytes were collected from 14 healthy volunteers at regular weekly intervals over 22 weeks. Vitamins were measured by HPLC. From the data generated, the intra- (CVI) and interindividual (CVG) biological CVs were estimated for each vitamin. Derived quality specifications, II, and RCV were calculated from CVI and CVG.

Results: CVI was 4.8%–38% and CVG was 10%–65% for the vitamins measured. The CVIs for vitamins A, E, B1, and B2 were lower (4.8%–7.6%) than for the other vitamins in blood. For all vitamins, CVG was higher than CVI, with II <1.0 (range, 0.36–0.95). The RCVs for vitamins were high (15.8%–108%). Apart from vitamins A, B1, and erythrocyte B2, the imprecision of our methods for measurement of vitamins in blood was within the desirable goal.

Conclusions: For most vitamin measurements in plasma, whole blood, or erythrocytes, the desirable imprecision goals based on biological variation are obtainable by current methodologies. Population reference intervals for vitamins are of limited value in demonstrating deficiency or excess.
Improving the levels of essential amino acids and sulfur metabolites in plants
Authors: Galili Gad , Amir Rachel , Hoefgen Rainer , Hesse Holger
Published: September 2005

Plants represent the major source of food for humans, either directly or indirectly through their use as livestock feeds. Plant foods are not nutritionally balanced because they contain low proportions of a number of essential metabolites, such as vitamins and amino acids, which humans and a significant proportion of their livestock cannot produce on their own. Among the essential amino acids needed in human diets, Lys, Met, Thr and Trp are considered as the most important because they are present in only low levels in plant foods. In the present review, we discuss approaches to improve the levels of the essential amino acids Lys and Met, as well as of sulfur metabolites, in plants using metabolic engineering approaches. We also focus on specific examples for which a deeper understanding of the regulation of metabolic networks in plants is needed for tailor-made improvements of amino acid metabolism with minimal interference in plant growth and productivity.
Developing an effective RNA interference strategy against a plus-strand RNA virus: silencing of coxsackievirus B3 and its cognate coxsackievirus-adenovirus receptor
Authors: Werk Denise , Schubert Steffen , Lindig Vanessa , Grunert Hans-Peter , Zeichhardt Heinz , Erdmann Volker A. , Kurreck Jens
Published: September 2005

Coxsackievirus B3 (CVB-3) is a plus-strand RNA virus that is believed to be the most common causal agent of viral myocarditis. Since no specific treatment for CVB-3 infections is available to date, we and others have recently started to develop RNA interference (RNAi) approaches to prevent virus propagation. Here we describe our strategy for the development of efficient small interfering RNAs (siRNAs) against viral genomes. Initially, fusion constructs of a reporter (green fluorescent protein) and viral subgenomic fragments were employed to select active siRNAs against the virus. Moreover, in an attempt to achieve sustained virus silencing and reduce the risk of generating escape mutants, only highly efficient siRNAs directed against regions of the viral genome that are unlikely to tolerate mutations were considered for virus inhibition. Two siRNAs directed against the 3D RNA-dependent RNA polymerase were found to inhibit virus propagation by 80–90%. The protective effect of the efficient siRNAs lasted for several days. Furthermore, we have first evidence that inhibition of the cellular coxsackievirus-adenovirus receptor (CAR) by RNAi also reduces the virus titre. Our strategy is likely to be applicable to other (RNA) viruses as well.
Regulation of asparagine synthetase gene transcription by the basic region leucine zipper transcription factors ATF5 and CHOP
Authors: Al Sarraj Jude , Vinson Charles , Thiel Gerald
Published: September 2005

Asparagine synthetase catalyses the glutamine- and ATP-dependent conversion of aspartic acid to asparagine. In human hepatoma cells cultured in mediumcontaining amino acids, the mRNA of asparagine synthetase is not detectable by RNase protection mapping. However, maintaining the cells in amino acid-free Krebs-Ringer bicarbonate buffer strongly upregulated asparagine synthetase biosynthesis. The effect of amino acid deprivation on asparagine synthetase gene transcription is mediated by a genetic element termed the nutrient-sensing response unit. Previous studies revealed that the basic region leucine zipper (bZIP) transcription factor CREB2/ATF4 is involved in the nutrient deprivation-induced upregulation of asparagine synthetase gene transcription. Here we show that overexpression of the bZIP protein ATF5, a transcriptional activator, stimulates asparagine synthetase promoter/reporter gene transcription via the nutrient-sensing response unit. In contrast, ATF5 does not transactivate cAMP response element (CRE)-containing reporter genes. Overexpression of the C/EBP homologous transcription factor CHOP impaired transcriptional activation of the asparagine synthetase promoter following amino acid deprivation or over-expression of ATF5 or CREB2/ATF4. These data indicate that CHOP functions as a shut-off-device for nutrient deprivation-induced gene transcription.
Cholesterol is the major component of native lipoproteins activating the p38 mitogen-activated protein kinases
Authors: Dobreva Iveta , Zschörnig Olaf , Waeber Gérard , James Richard W. , Widmann Christian
Published: September 2005

Elevated low-density lipoprotein (LDL) levels induce activation of the p38 mitogen-activated protein kinase (MAPK), a stress-activated protein kinase potentially participating in the development of atherosclerosis. The nature of the lipoprotein components inducing p38 MAPK activation has remained unclear however. We show here that both LDLs and high-density lipoproteins (HDLs) have the ability to stimulate the p38 MAPKs with potencies that correlate with their cholesterol content. Cholesterol solubilized in methyl-β-cyclodextrin was sufficient to activate the p38 MAPK pathway. Liposomes made of phosphatidylcholine (PC) or sphingomyelin, the two main phospholipids found in lipoproteins, were unable to stimulate the p38 MAPKs. In contrast, PC liposomes loaded with cholesterol potently activated this pathway. Reducing the cholesterol content of LDL particles lowered their ability to activate the p38 MAPKs. Cell lines representative of the three main cell types found in blood vessels (endothelial cells, smooth muscle cells and fibroblasts) all activated their p38 MAPK pathway in response to LDLs or cholesterol-loaded PC liposomes. These results indicate that elevated cholesterol content in lipoproteins, as seen in hypercholesterolemia, favors the activation of the stress-activated p38 MAPK pathway in cells from the vessel wall, an event that might contribute to the development of atherosclerosis.
Focal adhesion kinase is redistributed to focal complexes and mediates cell spreading in macrophages in response to M-CSF
Authors: Rovida Elisabetta , Lugli Benedetta , Barbetti Valentina , Giuntoli Serena , Olivotto Massimo , Dello Sbarba Persio
Published: September 2005

The macrophage colony-stimulating factor (M-CSF, CSF-1) regulates survival, proliferation and differentiation of mononuclear phagocytes, as well as macrophage motility and morphology. The latter features are usually regulated by ECM-mediated activation of integrins and subsequent tyrosine phosphorylation of cellular proteins, including focal adhesion kinase (FAK). FAK is phosphorylated by downstream receptor tyrosine kinases as well. We addressed the question whether M-CSF regulates FAK tyrosine phosphorylation in macrophages, and found that M-CSF induces FAK phosphorylation at all known tyrosine residues. This phosphorylation was dependent on Src. Extracellularly-regulated kinase (ERK), Jun N-terminal kinase (JNK) and phosphatidylinositol-3-kinase (PI3K) were found to be negatively involved in M-CSF-induced FAK phosphorylation, as their inhibition resulted in FAK hyper-phosphorylation. Following M-CSF treatment, FAK and the active forms of M-CSFR and Src were redistributed to the cytoskeleton, where active ERK, JNK and PI3K were detectable. Immunofluorescence showed the presence of FAK and its active form in focal complexes following M-CSF treatment. Moreover, cell spreading and adhesion were impaired when FAK tyrosine phosphorylation was abrogated by either transfection with FRNK, a dominant negative form of FAK, or treatment with a number of inhibitors of upstream FAK-activating signals. These results point to a relevant role for FAK in the regulation of cell spreading and adhesion in macrophages.
Activation processing of cathepsin H impairs recognition by its propeptide
Authors: Horn Martin , Dolečková-Marešová Lucie , Rulíšek Lubomír , Máša Martin , Vasiljeva Olga , Turk Boris , Gan-Erdene Tudeviin , Baudyš Miroslav , Mareš Michael
Published: September 2005

Free propeptides are known to function as inhibitors of the parental mature cysteine cathepsins. This general rule, however, does not apply to the aminopeptidase cathepsin H. Screening of propeptide fragments for their inhibitory potency revealed no significant effect on the native mature cathepsin H. On the other hand, inhibitory interaction was established with recombinant cathepsin H that displays endopeptidase activity due to a lack of the mini-chain. This finding suggests that the propeptide-binding region is structurally rearranged during maturation processing and mini-chain formation, which impairs the effective recognition of mature cathepsin H by its own propeptide.
Sino-Pakistan Relations and Xinjiang's Uighurs: Politics, Trade, and Islam along the Karakoram Highway
Authors: Haider Ziad
Published: September 2005

China's Muslim Uighurs have recently proven to be an unusual source of friction in the stalwart Sino-Pakistan friendship. This essay analyzes how politics in the Xinjiang Uighur Autonomous Region and trade and movement along the Karakoram Highway linking Xinjiang with Pakistan have affected the relationships among Beijing, Islamabad, the Uighurs, and the Pakistani traders operating in Xinjiang.
Russia's New Role in Central Asia
Authors: Buszynski Leszek
Published: September 2005

During the Yeltsin era, Russian interest in Central Asia lapsed. Under Vladimir Putin, Russia has moved to strengthen its role in the region because of Islamic fundamentalism, fear of displacement by America, and the presence of oil and natural gas reserves. The problem is that Putin has relied upon local authoritarian leaders for influence, which exposes Russia to regional political change.
Evaluating Nepal's Integrated “Security” and “Development” Policy: Development, Democracy, and Counterinsurgency
Authors: Gordon Stuart
Published: September 2005

Development, democratization, and security policies have a complex and often troubled relationship in Nepal. This article explores this relationship in terms of both the grievances underlying the insurgency and the government's overall security and development responses.
Regional Hegemonies and the Exercise of Power in Southeast Asia: A Study of Indonesia and Vietnam
Authors: Emmers Ralf
Published: September 2005

This article investigates efforts by Indonesia and Vietnam to achieve hegemony within their respective sub-regions of Southeast Asia. The two countries have exercised both benevolent and coercive power in their foreign policy. The article examines under which circumstances uses of benevolent and coercive instruments have helped the countries attain hegemony.