bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of hooking damage and hook type on post-release survival of sand flathead (Platycephalus bassensis)
Authors: Lyle Jeremy M. , Moltschaniwskyj Natalie A. , Morton Alastair J. , Brown Ian W. , Mayer David
Published: May 2007

This study examined post-release survival in sand flathead (Platycephalus bassensis) and whether there were survival benefits from the use of circle hooks over conventional hook patterns. Anatomical hooking location was the major factor contributing to mortality, with an almost 100% survival rate for fish hooked in the lip, mouth or eye (shallow-hooked) compared with around 64% for fish hooked in the throat or gut (deep-hooked). Mortality in deep-hooked fish was generally associated with injuries to vital organs (gills, heart, liver) and survival was significantly lower if bleeding was associated with injury (54% compared with 85% for non-bleeders). Circle hooks resulted in significantly lower deep-hooking rates (1%) compared with conventional hook types (4–9%) and, based on catch rates, were at least as effective as conventional hook patterns. Estimated survival rates for line-caught sand flathead were high, over 99% for circle hooks and between 94 and 97% for conventional hooks. These findings support the efficacy of management strategies based on size and bag limits and the practice of catch-and-release fishing for sand flathead, as well as a potential conservation benefit from the use of circle hooks.
The effects of turbidity and complex habitats on the feeding of a galaxiid fish are clear and simple
Authors: Stuart-Smith R. D. , Stuart-Smith J. F. , White R. W. G. , Barmuta L. A.
Published: May 2007

The habitat used by animals plays an important role in their interactions with predators and prey. By using complex habitats such as areas of dense macrophyte cover in response to elevated predation risk, small fishes may reduce their foraging success. Because the threat of predation by introduced brown trout increases the use of complex habitats by the threatened Galaxias auratus (Johnston), we experimentally examined its foraging in different habitats to estimate indirect impacts of brown trout presence. The lakes in which G. auratus lives have recently become more turbid, so the experiment was also conducted under different turbidity levels. Laboratory feeding trials in which planktonic and epibenthic prey were simultaneously offered to G. auratus in the presence or absence of artificial macrophytes and at three turbidity levels (0, 50 and 100 NTU) revealed that its overall foraging success was unaffected by habitat complexity; however, in trials with artificial macrophytes, G. auratus consumed a greater proportion of planktonic prey than in the absence of artificial macrophytes. Neither overall foraging success nor prey selection by G. auratus was affected by high turbidity, indicating that water clarity does not appear to directly negatively impact its feeding. The switch in prey types would probably not be detrimental to G. auratus in the long term, and thus it appears that there is no substantial feeding cost associated with its increased use of complex habitats. It could, however, affect lower trophic levels in the lakes to which it is endemic.
Effects of simulated green turtle regrazing on seagrass abundance, growth and nutritional status in Moreton Bay, south-east Queensland, Australia
Authors: Kuiper-Linley Marianne , Johnson Craig R. , Lanyon Janet M.
Published: May 2007

In some parts of their range, green turtles maintain grazing plots in seagrass beds by regular regrazing. The effects of simulated repeated grazing on subtropical seagrasses in Moreton Bay, Australia were investigated in a manipulative experiment over summer. Three seagrass species were subjected to two different clipping frequencies (simulating turtle cropping) and compared with unclipped controls over a 3.5-month summer period for the effects on seagrass biomass, leaf size and regrowth rates and water-soluble carbohydrate (WSC) and starch content. The order of the seagrass species’ relative tolerance to simulated grazing was Halophila ovalis > Zostera capricorni > Cymodocea serrulata. Frequent regrazing of the green turtle’s preferred seagrass, H. ovalis, resulted in an increase in leaf regrowth rate so that standing biomass of leaves and total plant material was maintained, suggesting an increase in productivity. Furthermore, whole-plant concentrations of WSC increased significantly in clipped H. ovalis plants relative to unclipped controls. In contrast, leaf biomass of the seagrass species less preferred by turtles, Z. capricorni and C. serrulata, decreased in response to repeated leaf removal relative to controls, despite maintenance of leaf regrowth rates. C. serrulata responded to repeated clipping with a reduction in leaf size and a decrease in rhizome WSC concentration. Z. capricorni also produced fewer and smaller new leaves. The ability of the preferred species, H. ovalis, to increase production of nutrient-rich standing crop in response to regrazing has major implications for green turtles and other seagrass grazers.
Common genetic variation within the Low-Density Lipoprotein Receptor-Related Protein 6 and late-onset Alzheimer's disease
Authors: De Ferrari Giancarlo V. , Papassotiropoulos Andreas , Biechele Travis , Wavrant De-Vrieze Fabienne , Avila Miguel E. , Major Michael B. , Myers Amanda , Sáez Katia , Henríquez Juan P. , Zhao Alice , Wollmer M. Axel , Nitsch Roger M. , Hock Christoph , Morris Chris M. , Hardy John , Moon Randall T.
Published: May 2007

Genome-wide linkage studies have defined a broad susceptibility region for late-onset Alzheimer's disease on chromosome 12, which contains the Low-Density Lipoprotein Receptor-Related Protein 6 (
LRP6
) gene, a coreceptor for Wnt signaling. Here, we report the association between common
LRP6
variants and late-onset Alzheimer's disease in a multicenter case-control series as well as in a large family-based series ascertained by the National Institute of Mental Health–National Institute on Aging Genetics Initiative. As shown in the genome-wide linkage studies, our association depends mainly on apolipoprotein E-ε4 (
APOE
-ε
4
) carrier status. Haplotype tagging single-nucleotide polymorphisms (SNPs) with a set of seven allelic variants of
LRP6
identified a putative risk haplotype, which includes a highly conserved coding sequence SNP: Ile-1062 → Val. Functional analyses revealed that the associated allele
Val-1062
, an allele previously linked to low bone mass, has decreased β-catenin signaling in HEK293T cells. Our study unveils a genetic relationship between
LRP6
and
APOE
and supports the hypothesis that altered Wnt/β-catenin signaling may be involved in this neurodegenerative disease.
Collapse of a fish population after exposure to a synthetic estrogen
Authors: Kidd Karen A. , Blanchfield Paul J. , Mills Kenneth H. , Palace Vince P. , Evans Robert E. , Lazorchak James M. , Flick Robert W.
Published: May 2007

Municipal wastewaters are a complex mixture containing estrogens and estrogen mimics that are known to affect the reproductive health of wild fishes. Male fishes downstream of some wastewater outfalls produce vitellogenin (VTG) (a protein normally synthesized by females during oocyte maturation) and early-stage eggs in their testes, and this feminization has been attributed to the presence of estrogenic substances such as natural estrogens [estrone or 17β-estradiol (E2)], the synthetic estrogen used in birth-control pills [17α-ethynylestradiol (EE2)], or weaker estrogen mimics such as nonylphenol in the water. Despite widespread evidence that male fishes are being feminized, it is not known whether these low-level, chronic exposures adversely impact the sustainability of wild populations. We conducted a 7-year, whole-lake experiment at the Experimental Lakes Area (ELA) in northwestern Ontario, Canada, and showed that chronic exposure of fathead minnow (
Pimephales promelas
) to low concentrations (5–6 ng·L
−1
) of the potent 17α-ethynylestradiol led to feminization of males through the production of vitellogenin mRNA and protein, impacts on gonadal development as evidenced by intersex in males and altered oogenesis in females, and, ultimately, a near extinction of this species from the lake. Our observations demonstrate that the concentrations of estrogens and their mimics observed in freshwaters can impact the sustainability of wild fish populations.
Threatening a rubber hand that you feel is yours elicits a cortical anxiety response
Authors: Ehrsson H. Henrik , Wiech Katja , Weiskopf Nikolaus , Dolan Raymond J. , Passingham Richard E.
Published: May 2007

The feeling of body ownership is a fundamental aspect of self-consciousness. The underlying neural mechanisms can be studied by using the illusion where a person is made to feel that a rubber hand is his or her own hand by brushing the person's hidden real hand and synchronously brushing the artificial hand that is in full view. Here we show that threat to the rubber hand can induce a similar level of activity in the brain areas associated with anxiety and interoceptive awareness (insula and anterior cingulate cortex) as when the person's real hand is threatened. We further show that the stronger the feeling of ownership of the artificial hand, the stronger the threat-evoked neuronal responses in the areas reflecting anxiety. Furthermore, across subjects, activity in multisensory areas reflecting ownership predicted the activity in the interoceptive system when the hand was under threat. Finally, we show that there is activity in medial wall motor areas, reflecting an urge to withdraw the artificial hand when it is under threat. These findings suggest that artificial limbs can evoke the same feelings as real limbs and provide objective neurophysiological evidence that the rubber hand is fully incorporated into the body. These findings are of fundamental importance because they suggest that the feeling of body ownership is associated with changes in the interoceptive systems.
Cryptic speciation in a model invertebrate chordate
Authors: Caputi Luigi , Andreakis Nikos , Mastrototaro Francesco , Cirino Paola , Vassillo Mauro , Sordino Paolo
Published: May 2007

We applied independent species concepts to clarify the phylogeographic structure of the ascidian
Ciona intestinalis
, a powerful model system in chordate biology and for comparative genomic studies. Intensive research with this marine invertebrate is based on the assumption that natural populations globally belong to a single species. Therefore, understanding the true taxonomic classification may have implications for experimental design and data management. Phylogenies inferred from mitochondrial and nuclear DNA markers accredit the existence of two cryptic species:
C. intestinalis
sp. A, genetically homogeneous, distributed in the Mediterranean, northeast Atlantic, and Pacific, and
C. intestinalis
sp. B, geographically structured and encountered in the North Atlantic. Species-level divergence is further entailed by cross-breeding estimates.
C. intestinalis
A and B from allopatric populations cross-fertilize, but hybrids remain infertile because of defective gametogenesis. Although anatomy illustrates an overall interspecific similarity lacking in diagnostic features, we provide consistent tools for in-field and in-laboratory species discrimination. Finding of two cryptic taxa in
C. intestinalis
raises interest in a new tunicate genome as a gateway to studies in speciation and ecological adaptation of chordates.
Inhibition of type I and type III interferons by a secreted glycoprotein from Yaba-like disease virus
Authors: Huang Jiaying , Smirnov Sergey V. , Lewis-Antes Anita , Balan Murugabaskar , Li Wei , Tang Sheila , Silke Gemma V. , Pütz Mike M. , Smith Geoffrey L. , Kotenko Sergei V.
Published: May 2007

Type I (IFN-α/β) and type III (IFN-λs) IFNs are important components of the host antiviral response. Although type III IFNs possess intrinsic antiviral activity similar to that of type I IFNs, they signal through a specific unique receptor complex, and their functional importance for antiviral resistance is largely uncharacterized. Here, we report the first virus defense mechanism that directly targets type III IFNs. Y136 from Yaba-like disease virus, a yatapoxvirus, is a secreted glycoprotein related to protein B18 from
Vaccinia
virus, a known type I IFN-binding protein and a member of the Ig superfamily. Surprisingly, whereas B18 inhibits only type I IFNs, Y136 inhibits both type I and type III IFNs. Y136 inhibits IFN-induced signaling and suppresses IFN-mediated biological activities including up-regulation of MHC class I antigen expression and induction of the antiviral state. These data demonstrate that poxviruses have developed unique strategies to counteract IFN-mediated antiviral protection and highlight the importance of type III IFNs in antiviral defense. These results suggest that type III IFNs may be an effective treatment for some poxviral infections.
Functional neuroimaging and behavioral correlates of capacity decline in visual short-term memory after sleep deprivation
Authors: Chee Michael W. L. , Chuah Y. M. Lisa
Published: May 2007

Sleep deprivation (SD) impairs short-term memory, but it is unclear whether this is because of reduced storage capacity or processes contributing to appropriate information encoding. We evaluated 30 individuals twice, once after a night of normal sleep and again after 24 h of SD. In each session, we evaluated visual memory capacity by presenting arrays of one to eight colored squares. Additionally, we measured cortical responses to varying visual array sizes without engaging memory. The magnitude of intraparietal sulcus activation and memory capacity after normal sleep were highly correlated. SD elicited a pattern of activation in both tasks, indicating that deficits in visual processing and visual attention accompany and could account for loss of short-term memory capacity. Additionally, a comparison between better and poorer performers showed that preservation of precuneus and temporoparietal junction deactivation with increasing memory load corresponds to less performance decline when one is sleep-deprived.
Structural insight into dimeric interaction of the SARAH domains from Mst1 and RASSF family proteins in the apoptosis pathway
Authors: Hwang Eunha , Ryu Kyoung-Seok , Pääkkönen Kimmo , Güntert Peter , Cheong Hae-Kap , Lim Dae-Sik , Lee Jie-Oh , Jeon Young Ho , Cheong Chaejoon
Published: May 2007

In eukaryotic cells, apoptosis and cell cycle arrest by the Ras → RASSF → MST pathway are controlled by the interaction of SARAH (for Salvador/Rassf/Hippo) domains in the C-terminal part of tumor suppressor proteins. The Mst1 SARAH domain interacts with its homologous domain of Rassf1 and Rassf5 (also known as Nore1) by forming a heterodimer that mediates the apoptosis process. Here, we describe the homodimeric structure of the human Mst1 SARAH domain and its heterotypic interaction with the Rassf5 and Salvador (Sav) SARAH domain. The Mst1 SARAH structure forms a homodimer containing two helices per monomer. An antiparallel arrangement of the long α-helices (h2/h2′) provides an elongated binding interface between the two monomers, and the short 3
10
helices (h1/h1′) are folded toward that of the other monomer. Chemical shift perturbation experiments identified an elongated, tight-binding interface with the Rassf5 SARAH domain and a 1:1 heterodimer formation. The linker region between the kinase and the SARAH domain is shown to be disordered in the free protein. These results imply a novel mode of interaction with RASSF family proteins and provide insight into the mechanism of apoptosis control by the SARAH domain.
Niche characteristics explain the reciprocal invasion success of stream salmonids in different continents
Authors: Korsu Kai , Huusko Ari , Muotka Timo
Published: May 2007

An ability to understand and predict invasions is elemental for controlling the detrimental effects of introduced organisms on native biota. In eastern North America, European brown trout generally dominates over, and eventually replaces, the native brook trout. We show here that in northern Europe the pattern of replacement between these two species is reversed: when transferred to North European streams, brook trout spread extensively and partially replaced the native brown trout. The effect of brook trout on brown trout was habitat-specific: brook trout excluded the native species only in small tributary streams where the reproduction of brown trout was severely reduced, whereas in larger streams brown trout was largely unaffected. Thus, the pattern of coexistence among the two salmonids in our study area is approaching that typically observed in North American streams. In both areas, brook trout ultimately settles in small headwater streams, but the process of replacement differs profoundly: in North Europe, brook trout replaces brown trout in headwater streams, whereas in North America these same streams are the ultimate refuge area for brook trout under the invasion pressure by brown trout. Our results underline the importance of knowing species' niche characteristics to explain and predict biological invasions.
Experimental observation of quantum oscillation of surface chemical reactivities
Authors: Ma Xucun , Jiang Peng , Qi Yun , Jia Jinfeng , Yang Yu , Duan Wenhui , Li Wei-Xue , Bao Xinhe , Zhang S. B. , Xue Qi-Kun
Published: May 2007

Here we present direct observation of a quantum reactivity with respect to the amounts of O
2
adsorbed and the rates of surface oxidation as a function of film thickness on ultrathin (2–6 nm) Pb mesas by scanning tunneling microscopy. Simultaneous spectroscopic measurements on the electronic structures reveal a quantum oscillation that originates from quantum well states of the mesas, as a generalization of the Fabry–Pérot modes of confined electron waves. We expect the quantum reactivity to be a general phenomenon for most ultrathin metal films with broad implications, such as nanostructure tuning of surface reactivities and rational design of heterogeneous catalysts.
Characterization of actomyosin bond properties in intact skeletal muscle by force spectroscopy
Authors: Colombini Barbara , Bagni M. Angela , Romano Giovanni , Cecchi Giovanni
Published: May 2007

Force generation and motion in skeletal muscle result from interaction between actin and myosin myofilaments through the cyclical formation and rupture of the actomyosin bonds, the cross-bridges, in the overlap region of the sarcomeres. Actomyosin bond properties were investigated here in single intact muscle fibers by using dynamic force spectroscopy. The force needed to forcibly detach the cross-bridge ensemble in the half-sarcomere (hs) was measured in a range of stretching velocity between 3.4 × 10
3
nm·hs
−1
·s
−1
or 3.3 fiber length per second (
l
0
s
−1
) and 6.1 × 10
4
nm·hs
−1
·s
−1
or 50
l
0
·s
−1
during tetanic force development. The rupture force of the actomyosin bond increased linearly with the logarithm of the loading rate, in agreement with previous experiments on noncovalent single bond and with Bell theory [Bell GI (1978)
Science
200:618–627]. The analysis permitted calculation of the actomyosin interaction length,
x
β
and the dissociation rate constant for zero external load,
k
0
. Mean
x
β
was 1.25 nm, a value similar to that reported for single actomyosin bond under rigor condition. Mean
k
0
was 20 s
−1
, a value about twice as great as that reported in the literature for isometric force relaxation in the same type of muscle fibers. These experiments show, for the first time, that force spectroscopy can be used to reveal the properties of the individual cross-bridge in intact skeletal muscle fibers.
Heat shock-inducible Cre/Lox approaches to induce diverse types of tumors and hyperplasia in transgenic zebrafish
Authors: Le Xiuning , Langenau David M. , Keefe Matthew D. , Kutok Jeffery L. , Neuberg Donna S. , Zon Leonard I.
Published: May 2007

RAS family members are among the most frequently mutated oncogenes in human cancers. Given the utility of zebrafish in both chemical and genetic screens, developing RAS-induced cancer models will make large-scale screens possible to understand further the molecular mechanisms underlying malignancy. We developed a heat shock-inducible Cre/Lox-mediated transgenic approach in which activated human
kRASG12D
can be conditionally induced within transgenic animals by heat shock treatment. Specifically, double transgenic fish Tg(
B-actin-LoxP-EGFP-LoxP-kRASG12D; hsp70-Cre
) developed four types of tumors and hyperplasia after heat shock of whole zebrafish embryos, including rhabdomyosarcoma, myeloproliferative disorder, intestinal hyperplasia, and malignant peripheral nerve sheath tumor. Using
ex vivo
heat shock and transplantation of whole kidney marrow cells from double transgenic animals, we were able to generate specifically
kRASG12D
-induced myeloproliferative disorder in recipient fish. This heat shock-inducible recombination approach allowed for the generation of multiple types of RAS-induced tumors and hyperplasia without characterizing tissue-specific promoters. Moreover, these tumors and hyperplasia closely resemble human diseases at both the morphologic and molecular levels.
Primary protein response after ligand photodissociation in carbonmonoxy myoglobin
Authors: Sato Akira , Gao Ying , Kitagawa Teizo , Mizutani Yasuhisa
Published: May 2007

Time-resolved UV resonance Raman (UVRR) spectroscopic studies of WT and mutant myoglobin were performed to reveal the dynamics of protein motion after ligand dissociation. After dissociation of carbon monoxide (CO) from the heme, UVRR bands of Tyr showed a decrease in intensity with a time constant of 2 ps. The intensity decrease was followed by intensity recovery with a time constant of 8 ps. On the other hand, UVRR bands of Trp residues located in the A helix showed an intensity decrease that was completed within the instrument response time. The intensity decrease was followed by an intensity recovery with a time constant of ≈50 ps and lasted up to 1 ns. The time-resolved UVRR study of the myoglobin mutants demonstrated that the hydrophobicity of environments around Trp-14 decreased, whereas that around Trp-7 barely changed in the primary protein response. The present data indicate that displacement of the E helix toward the heme occurs within the instrument response time and that movement of the FG corner takes place with a time constant of 2 ps. The finding that the instantaneous motion of the E helix strongly suggests a mechanism in which protein structural changes are propagated from the heme to the A helix through the E helix motion.
Single-molecule force spectroscopy reveals a mechanically stable protein fold and the rational tuning of its mechanical stability
Authors: Sharma Deepak , Perisic Ognjen , Peng Qing , Cao Yi , Lam Canaan , Lu Hui , Li Hongbin
Published: May 2007

It is recognized that shear topology of two directly connected force-bearing terminal β-strands is a common feature among the vast majority of mechanically stable proteins known so far. However, these proteins belong to only two distinct protein folds, Ig-like β sandwich fold and β-grasp fold, significantly hindering delineating molecular determinants of mechanical stability and rational tuning of mechanical properties. Here we combine single-molecule atomic force microscopy and steered molecular dynamics simulation to reveal that the
de novo
designed Top7 fold [Kuhlman B, Dantas G, Ireton GC, Varani G, Stoddard BL, Baker D (2003)
Science
302:1364–1368] represents a mechanically stable protein fold that is distinct from Ig-like β sandwich and β-grasp folds. Although the two force-bearing β strands of Top7 are not directly connected, Top7 displays significant mechanical stability, demonstrating that the direct connectivity of force-bearing β strands in shear topology is not mandatory for mechanical stability. This finding broadens our understanding of the design of mechanically stable proteins and expands the protein fold space where mechanically stable proteins can be screened. Moreover, our results revealed a substructure-sliding mechanism for the mechanical unfolding of Top7 and the existence of two possible unfolding pathways with different height of energy barrier. Such insights enabled us to rationally tune the mechanical stability of Top7 by redesigning its mechanical unfolding pathway. Our study demonstrates that computational biology methods (including
de novo
design) offer great potential for designing proteins of defined topology to achieve significant and tunable mechanical properties in a rational and systematic fashion.
Evidence for emergence of diverse polioviruses from C-cluster coxsackie A viruses and implications for global poliovirus eradication
Authors: Jiang Ping , Faase Johan A. J. , Toyoda Hidemi , Paul Aniko , Wimmer Eckard , Gorbalenya Alexander E.
Published: May 2007

The poliovirus (PV) eradication campaign is conducted on the premise that this virus, because of the lack of a zoonotic reservoir, will not reemerge once eradicated. This report examines the origin of PV using theoretical and experimental approaches. Our rooted phylogenetic analysis suggests a speciation of PV from a C-cluster coxsackie A virus (C-CAV) ancestor through mutation of the capsid that caused a receptor switch from intercellular adhesion molecule-1 to CD155, leading to a change of pathogenicity. This hypothesis is supported experimentally with chimeras generated from three different pairs of PV and C-CAV. Those carrying the PV capsid and the replication proteins of C-CAVs replicated well, whereas their reciprocal counterparts were either debilitated or dead. This phenomenon of asymmetry is observed also in recombinants between PV1 and C-CAV20, selected in tissue culture cells using a previously undescribed protocol. The recombinants are generated at frequencies of 10
−6
typical for PV interserotype recombination. Strikingly, they resemble genetically and phenotypically, including neurovirulence in
CD155
transgenic mice, the large majority of circulating vaccine-derived PVs that have caused poliomyelitis outbreaks in different parts of the world. These data provide experimental evidence for C-CAVs being partners to PVs in generating diverse PV progeny by homologous recombination. They support speciation of a novel human pathogen (PV) from a pool of different human pathogens (C-CAVs). In a PV-free world without PV neutralizing antibodies, contemporary C-CAV, like their ancestor(s), could be fertile ground for a PV-like agent to emerge by mutation.
Sequential structures provide insights into the fidelity of RNA replication
Authors: Ferrer-Orta Cristina , Arias Armando , Pérez-Luque Rosa , Escarmís Cristina , Domingo Esteban , Verdaguer Nuria
Published: May 2007

RNA virus replication is an error-prone event caused by the low fidelity of viral RNA-dependent RNA polymerases. Replication fidelity can be decreased further by the use of mutagenic ribonucleoside analogs to a point where viral genetic information can no longer be maintained. For foot-and-mouth disease virus, the antiviral analogs ribavirin and 5-fluorouracil have been shown to be mutagenic, contributing to virus extinction through lethal mutagenesis. Here, we report the x-ray structure of four elongation complexes of foot-and-mouth disease virus polymerase 3D obtained in presence of natural substrates, ATP and UTP, or mutagenic nucleotides, ribavirin triphosphate and 5-fluorouridine triphosphate with different RNAs as template–primer molecules. The ability of these complexes to synthesize RNA in crystals allowed us to capture different successive replication events and to define the critical amino acids involved in (
i
) the recognition and positioning of the incoming nucleotide or analog; (
ii
) the positioning of the acceptor base of the template strand; and (
iii
) the positioning of the 3′-OH group of the primer nucleotide during RNA replication. The structures identify key interactions involved in viral RNA replication and provide insights into the molecular basis of the low fidelity of viral RNA polymerases.
In situ
measurement of the electrical potential across the phagosomal membrane using FRET and its contribution to the proton-motive force
Authors: Steinberg Benjamin E. , Touret Nicolas , Vargas-Caballero Mariana , Grinstein Sergio
Published: May 2007

Phagosomes employ lytic enzymes, cationic peptides, and reactive oxygen intermediates to eliminate invading microorganisms. The effectiveness of these microbicidal mechanisms is potentiated by the acidic pH created by H
+
-pumping vacuolar-type ATPases (V-ATPases) on the phagosomal membrane. The degree of phagosomal acidification varies greatly among neutrophils, macrophages, and dendritic cells and can be affected by diseases like cystic fibrosis. The determinants of phagosomal pH are not completely understood, but the permeability to ions that neutralize the electrogenic effect of the V-ATPase has been proposed to play a central role. When counterion conductance is limiting, generation of a large membrane potential will dominate the proton-motive force (
pmf
), with a proportionally diminished pH gradient. Validation of this notion requires direct measurement of the electrical potential that develops across the phagosomal membrane (Ψ
Φ
). We describe a noninvasive procedure to estimate Ψ
Φ
in intact cells, based on fluorescence resonance energy transfer. This approach, in combination with measurements of phagosomal pH, enabled us to calculate the
pmf
across phagosomes of murine macrophages and to analyze the factors that limit acidification. At steady state, Ψ
Φ
averaged 27 mV (lumen positive) and was only partially dissipated by inhibition of the V-ATPase with concanamycin A. The comparatively small contribution of the potential to the
pmf
suggests that proton pumping is not limited by the counterion permeability, a notion that was validated independently by using ionophores. Instead, phagosomal pH stabilizes when the rate of proton pumping, which decreases gradually as the lumen acidifies, is matched by the passive leak of proton equivalents.
Polycystic disease caused by deficiency in xylosyltransferase 2, an initiating enzyme of glycosaminoglycan biosynthesis
Authors: Condac Eduard , Silasi-Mansat Robert , Kosanke Stanley , Schoeb Trenton , Towner Rheal , Lupu Florea , Cummings Richard D. , Hinsdale Myron E.
Published: May 2007

The basic biochemical mechanisms underlying many heritable human polycystic diseases are unknown despite evidence that most cases are caused by mutations in members of several protein families, the most prominent being the polycystin gene family, whose products are found on the primary cilia, or due to mutations in posttranslational processing and transport. Inherited polycystic kidney disease, the most prevalent polycystic disease, currently affects ≈500,000 people in the United States. Decreases in proteoglycans (PGs) have been found in tissues and cultured cells from patients who suffer from autosomal dominant polycystic kidney disease, and this PG decrease has been hypothesized to be responsible for cystogenesis. This is possible because alterations in PG concentrations would be predicted to disrupt many homeostatic mechanisms of growth, development, and metabolism. To test this hypothesis, we have generated mice lacking xylosyltransferase 2 (XylT2), an enzyme involved in PG biosynthesis. Here we show that inactivation of XylT2 results in a substantial reduction in PGs and a phenotype characteristic of many aspects of polycystic liver and kidney disease, including biliary epithelial cysts, renal tubule dilation, organ fibrosis, and basement membrane abnormalities. Our findings demonstrate that alterations in PG concentrations can occur due to loss of XylT2, and that reduced PGs can induce cyst development.
The origin of modern metabolic networks inferred from phylogenomic analysis of protein architecture
Authors: Caetano-Anollés Gustavo , Kim Hee Shin , Mittenthal Jay E.
Published: May 2007

Metabolism represents a complex collection of enzymatic reactions and transport processes that convert metabolites into molecules capable of supporting cellular life. Here we explore the origins and evolution of modern metabolism. Using phylogenomic information linked to the structure of metabolic enzymes, we sort out recruitment processes and discover that most enzymatic activities were associated with the nine most ancient and widely distributed protein fold architectures. An analysis of newly discovered functions showed enzymatic diversification occurred early, during the onset of the modern protein world. Most importantly, phylogenetic reconstruction exercises and other evidence suggest strongly that metabolism originated in enzymes with the P-loop hydrolase fold in nucleotide metabolism, probably in pathways linked to the purine metabolic subnetwork. Consequently, the first enzymatic takeover of an ancient biochemistry or prebiotic chemistry was related to the synthesis of nucleotides for the RNA world.
Gram-positive three-component antimicrobial peptide-sensing system
Authors: Li Min , Lai Yuping , Villaruz Amer E. , Cha David J. , Sturdevant Daniel E. , Otto Michael
Published: May 2007

To survive during colonization or infection of the human body, microorganisms must circumvent mechanisms of innate host defense. Antimicrobial peptides represent a key component of innate host defense, especially in phagocytes and on epithelial surfaces. However, it is not known how the clinically important group of Gram-positive bacteria sense antimicrobial peptides to coordinate a directed defensive response. By determining the genome-wide gene regulatory response to human β-defensin 3 in the nosocomial pathogen
Staphylococcus epidermidis
, we discovered an antimicrobial peptide sensor system that controls major specific resistance mechanisms of Gram-positive bacteria and is unrelated to the Gram-negative PhoP/PhoQ system. It contains a classical two-component signal transducer and an unusual third protein, all of which are indispensable for signal transduction and antimicrobial peptide resistance. Furthermore, our data indicate that a very short, extracellular loop with a high density of negative charges in the sensor protein is responsible for antimicrobial peptide binding and the observed specificity for cationic antimicrobial peptides. Our study shows that Gram-positive bacteria have developed an efficient and unique way of controlling resistance mechanisms to antimicrobial peptides, which may provide a promising target for antimicrobial drug development.
Tropical birds have a slow pace of life
Authors: Wiersma Popko , Muñoz-Garcia Agustí , Walker Amy , Williams Joseph B.
Published: May 2007

Tropical birds are relatively long-lived and produce few offspring, which develop slowly and mature relatively late in life, the slow end of the life-history axis, whereas temperate birds lie at the opposite end of this continuum. We tested the hypothesis that tropical birds have evolved a reduced basal metabolic rate (BMR). We measured BMR of 69 species of tropical birds, the largest data set amassed on metabolic rates of tropical birds, and compared these measurements with 59 estimates of BMR for temperate birds. Our analyses included conventional least squares regression, regressions based on phylogenetic independent contrasts, and a comparison of BMR of 13 phylogenetically matched pairs, one species from the tropics and one from northerly temperate areas. Our triptych showed that tropical birds had a reduced BMR, compelling evidence for a connection between the life history of tropical birds and a slow pace of life. Further, tropical migrants breeding in temperate habitats had a lower BMR than did temperate residents, suggesting that these migrants have physiological traits consistent with a slow pace of life. In addition, we determined that tropical birds had a lower cold-induced peak metabolic rate and thermogenic metabolic scope than temperate species, a finding that is consistent with the hypothesis that their environment has not selected for high levels of thermogenesis, or alternatively, that a slow pace of life may be incompatible with high thermogenic capacity. We conclude that physiological function correlates with the suite of life-history traits.
A new paradigm for electrostatic catalysis of radical reactions in vitamin B
12
enzymes
Authors: Sharma Pankaz K. , Chu Zhen T. , Olsson Mats H. M. , Warshel Arieh
Published: May 2007

The catalytic power of enzymes containing coenzyme B
12
cofactor has been, in some respects, the “last bastion” for the strain hypothesis. The present work explores the origin of this effect by using simulation methods that overcome the sampling difficulties of previous energy minimization studies. It is found that the major part of the catalytic effect is due to the electrostatic interaction between the ribose and the protein, and that the strain contribution is very small. Remarkably, enzymes can use electrostatic effects even in a radical process, when the charge distribution of the reacting fragments does not change significantly during the reaction. Electrostatic catalysis can, in such cases, be obtained by attaching a polar group to the leaving fragment and designing an active site that interacts more strongly with this group in the product state than in the reactant state. The finding that evolution had to use this trick provides further evidence to the observation that it is extremely hard to catalyze enzymatic reactions by nonelectrostatic factors. The trick used by B
12
enzymes may, in fact, be a very powerful new strategy in enzyme design.
Nanoprobes with near-infrared persistent luminescence for
in vivo
imaging
Authors: le Masne de Chermont Quentin , Chanéac Corinne , Seguin Johanne , Pellé Fabienne , Maîtrejean Serge , Jolivet Jean-Pierre , Gourier Didier , Bessodes Michel , Scherman Daniel
Published: May 2007

Fluorescence is increasingly used for
in vivo
imaging and has provided remarkable results. Yet this technique presents several limitations, especially due to tissue autofluorescence under external illumination and weak tissue penetration of low wavelength excitation light. We have developed an alternative optical imaging technique by using persistent luminescent nanoparticles suitable for small animal imaging. These nanoparticles can be excited before injection, and their
in vivo
distribution can be followed in real-time for more than 1 h without the need for any external illumination source. Chemical modification of the nanoparticles' surface led to lung or liver targeting or to long-lasting blood circulation. Tumor mass could also be identified on a mouse model.
Live-cell imaging reveals sequential oligomerization and local plasma membrane targeting of stromal interaction molecule 1 after Ca
2+
store depletion
Authors: Liou Jen , Fivaz Marc , Inoue Takanari , Meyer Tobias
Published: May 2007

Stromal interaction molecule 1 (STIM1) has recently been identified by our group and others as an endoplasmic reticulum (ER) Ca
2+
sensor that responds to ER Ca
2+
store depletion and activates Ca
2+
channels in the plasma membrane (PM). The molecular mechanism by which STIM1 transduces signals from the ER lumen to the PM is not yet understood. Here we developed a live-cell FRET approach and show that STIM1 forms oligomers within 5 s after Ca
2+
store depletion. These oligomers rapidly dissociated when ER Ca
2+
stores were refilled. We further show that STIM1 formed oligomers before its translocation within the ER network to ER–PM junctions. A mutant STIM1 lacking the C-terminal polybasic PM-targeting motif oligomerized after Ca
2+
store depletion but failed to form puncta at ER–PM junctions. Using fluorescence recovery after photobleaching measurements to monitor STIM1 mobility, we show that STIM1 oligomers translocate on average only 2 μm to reach ER–PM junctions, arguing that STIM1 ER-to-PM signaling is a local process that is suitable for generating cytosolic Ca
2+
gradients. Together, our live-cell measurements dissect the STIM1 ER-to-PM signaling relay into four sequential steps: (
i
) dissociation of Ca
2+
, (
ii
) rapid oligomerization, (
iii
) spatially restricted translocation to nearby ER–PM junctions, and (
iv
) activation of PM Ca
2+
channels.
Liposomal vaccines with conformation-specific amyloid peptide antigens define immune response and efficacy in APP transgenic mice
Authors: Muhs Andreas , Hickman David T. , Pihlgren Maria , Chuard Nathalie , Giriens Valérie , Meerschman Carine , van der Auwera Ingrid , van Leuven Fred , Sugawara Masae , Weingertner Marie-Catherine , Bechinger Burkhard , Greferath Ruth , Kolonko Nadine , Nagel-Steger Luitgard , Riesner Detlev , Brady Roscoe O. , Pfeifer Andrea , Nicolau Claude
Published: May 2007

We investigated the therapeutic effects of two different versions of Aβ1–15 (16)liposome-based vaccines. Inoculation of APP-V717IxPS-1 (APPxPS-1) double-transgenic mice with tetra-palmitoylated amyloid 1–15 peptide (palmAβ1–15), or with amyloid 1–16 peptide (PEG-Aβ1–16) linked to a polyethyleneglycol spacer at each end, and embedded within a liposome membrane, elicited fast immune responses with identical binding epitopes. PalmAβ1–15liposomal vaccine elicited an immune response that restored the memory defect of the mice, whereas that of PEG-Aβ1–16had no such effect. Immunoglobulins that were generated were predominantly of the IgG class with palmAβ1–15, whereas those elicited by PEG-Aβ1–16were primarily of the IgM class. The IgG subclasses of the antibodies generated by both vaccines were mostly IgG2b indicating noninflammatory Th2 isotype. CD and NMR revealed predominantly β-sheet conformation of palmAβ1–15and random coil of PEG-Aβ1–16. We conclude that the association with liposomes induced a variation of the immunogenic structures and thereby different immunogenicities. This finding supports the hypothesis that Alzheimer's disease is a “conformational” disease, implying that antibodies against amyloid sequences in the β-sheet conformation are preferred as potential therapeutic agents.
Pph3–Psy2 is a phosphatase complex required for Rad53 dephosphorylation and replication fork restart during recovery from DNA damage
Authors: O'Neill Bryan M. , Szyjka Shawn J. , Lis Ewa T. , Bailey Aaron O. , Yates John R. , Aparicio Oscar M. , Romesberg Floyd E.
Published: May 2007

Activation of the checkpoint kinase Rad53 is a critical response to DNA damage that results in stabilization of stalled replication forks, inhibition of late-origin initiation, up-regulation of dNTP levels, and delayed entry to mitosis. Activation of Rad53 is well understood and involves phosphorylation by the protein kinases Mec1 and Tel1 as well as in trans autophosphorylation by Rad53 itself. However, deactivation of Rad53, which must occur to allow the cell to recover from checkpoint arrest, is not well understood. Here, we present genetic and biochemical evidence that the type 2A-like protein phosphatase Pph3 forms a complex with Psy2 (Pph3–Psy2) that binds and dephosphorylates activated Rad53 during treatment with, and recovery from, methylmethane sulfonate-mediated DNA damage. In the absence of Pph3–Psy2, Rad53 dephosphorylation and the resumption of DNA synthesis are delayed during recovery from DNA damage. This delay in DNA synthesis reflects a failure to restart stalled replication forks, whereas, remarkably, genome replication is eventually completed by initiating late origins of replication despite the presence of hyperphosphorylated Rad53. These findings suggest that Rad53 regulates replication fork restart and initiation of late firing origins independently and that regulation of these processes is mediated by specific Rad53 phosphatases.
Functional characterization of human PFTK1 as a cyclin-dependent kinase
Authors: Shu Fang , Lv Shun , Qin Yan , Ma Xinlu , Wang Xin , Peng Xiaozhong , Luo Ying , Xu Bing-e , Sun Xiaoqing , Wu Jun
Published: May 2007

Cyclin-dependent kinases (CDKs) are crucial regulators of the eukaryotic cell cycle whose activities are controlled by associated cyclins. PFTK1 shares limited homology to CDKs, but its ability to associate with any cyclins and its biological functions remain largely unknown. Here, we report the functional characterization of human PFTK1 as a CDK. PFTK1 specifically interacted with cyclin D3 (CCND3) and formed a ternary complex with the cell cycle inhibitor p21
Cip1
in mammalian cells. We demonstrated that the kinase activity of PFTK1 depended on CCND3 and was negatively regulated by p21
Cip1
. Moreover, we identified the tumor suppressor Rb as a potential downstream substrate for the PFTK1/CCND3 complex. Importantly, knocking down PFTK1 expression by using siRNA caused cell cycle arrest at G
1
, whereas ectopic expression of PFTK1 promoted cell proliferation. Taken together, our data strongly suggest that PFTK1 acts as a CDK that regulates cell cycle progression and cell proliferation.
High-resolution scanning tunneling microscopy imaging of mesoscopic graphene sheets on an insulating surface
Authors: Stolyarova Elena , Rim Kwang Taeg , Ryu Sunmin , Maultzsch Janina , Kim Philip , Brus Louis E. , Heinz Tony F. , Hybertsen Mark S. , Flynn George W.
Published: May 2007

We present scanning tunneling microscopy (STM) images of single-layer graphene crystals examined under ultrahigh vacuum conditions. The samples, with lateral dimensions on the micrometer scale, were prepared on a silicon dioxide surface by direct exfoliation of crystalline graphite. The single-layer films were identified by using Raman spectroscopy. Topographic images of single-layer samples display the honeycomb structure expected for the full hexagonal symmetry of an isolated graphene monolayer. The absence of observable defects in the STM images is indicative of the high quality of these films. Crystals composed of a few layers of graphene also were examined. They exhibited dramatically different STM topography, displaying the reduced threefold symmetry characteristic of the surface of bulk graphite.
Protection of the glutamate pool concentration in enteric bacteria
Authors: Yan Dalai
Published: May 2007

The central nitrogen metabolic circuit in enteric bacteria consists of three enzymes: glutamine synthetase, glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH). With the carbon skeleton provided by 2-oxoglutarate, ammonia/ammonium (NH
4
+
) is assimilated into two central nitrogen intermediates, glutamate and glutamine. Although both serve as nitrogen donors for all biosynthetic needs, glutamate and glutamine play different roles. Internal glutamine serves as a sensor of external nitrogen availability, and its pool concentration decreases upon nitrogen limitation. A high glutamate pool concentration is required to maintain the internal K
+
pool. The configuration of high glutamate and low glutamine pools was disrupted in GOGAT
−
mutants under low NH
4
+
conditions: the glutamate pool was low, the difference between glutamate and glutamine was diminished, and growth was defective. When a GOGAT
−
mutant was cultured in an NH
4
+
-limited chemostat, two sequential spontaneous mutations occurred. Each resulted in a suppressor mutant that outgrew its predecessor in the chemostat. The first suppressor overexpressed GDH, and the second also had a partially impaired glutamine synthetase. The result was a triple mutant in which NH
4
+
was assimilated by two enzymes instead of the normal three and yet glutamate and glutamine pools and growth were essentially normal. The results indicate preference for the usual ratio of glutamate and glutamine and the resilient and compensatory nature of the circuit on pool control. Analysis of other suppressor mutants selected on solid medium suggests that increased GDH expression is the key for rescue of the growth defect of GOGAT
−
mutants under low NH
4
+
conditions.