bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of behaviour and ontogeny on the locomotor performance of a West Indian lizard, Anolis lineatopus
Authors: Irschick D. J.
Published: March 2003

1. The speeds of locomotion in nature were quantified for juveniles and adult females of the lizard Anolis lineatopus during escape from a threat, feeding and undisturbed locomotion (juveniles only), and compared these data both with data gathered previously on maximal laboratory performance, and with field data gathered previously for adult males.

2. The following questions were addressed: (1) Within a particular intraspecific class, do lizards move at different speeds during different behaviours? (2) Within a particular behaviour, do intraspecific classes differ in speed? (3) Do juveniles differ from adult males in the entire distribution of speeds and distances moved when undisturbed?

3. Locomotion was typically fastest during escape, followed by feeding and then undisturbed locomotion. Within escape and undisturbed activity, the intraspecific classes differed significantly in absolute speed, relative speed and percentage maximal speed. For the entire distribution of movements during undisturbed locomotion, juveniles moved shorter distances and at slower speeds (between 0 and 10% of maximal) than adult males.

4. The relatively high percentages of maximal speed that juveniles employ when escaping and feeding provide limited support for the hypothesis that juveniles are compensating for their relatively poor sprinting capacities.
Seasonal decline in male‐phase duration in a protandrous plant: a response to increased mating opportunities?
Authors: Sargent R. D. , Roitberg B. D.
Published: March 2003

1. We examined the effects of pollinator visitation and time of season on male‐ and female‐phase duration, using experimental manipulation and survey data from naturally occurring populations of Chamerion (= Epilobium) angustifolium (L.) J. Holub (Onagraceae).

2. Based on the observation that male mating opportunity (numbers of female flowers/numbers of male flowers) increases seasonally, we predicted that individual flowers should spend more time in the male phase early in the season when mating opportunity is low. We predicted that if seasonal changes in mating opportunity select for phase duration, male‐phase duration should decline when pollinator effects are experimentally controlled.

3. A comparison of phase duration in naturally pollinated and pollinator‐excluded plants supported this prediction: male‐phase duration in the pollinator‐exclusion treatment was longer and declined faster than in the naturally pollinated group.

4. A population survey revealed that once the effects of temperature were controlled for, male‐phase duration was negatively correlated with date, while female‐phase duration was positively correlated with date.

5. These findings suggest that seasonal variation in mating opportunity, and not just pollination rate or temperature, may play a significant role in phase duration in dichogamous plants.
Crown carbon gain and elevated [CO2] responses of understorey saplings with differing allometry and architecture
Authors: E. Naumburg , D. S. Ellsworth , R. W. Pearcy
Published: March 2003

1. Attempts at determining the physiological basis of species’ differences, such as the ability to grow in deep shade, have been of limited success. However, this basis is fundamental to predicting species’ responses to rising atmospheric CO2 in the forest understorey. We linked a leaf photosynthesis and a tree architecture model to predict the effects of dynamic and steady state photosynthetic characteristics, crown architecture and elevated atmospheric CO2 concentration ([CO2]) on crown‐level carbon gain (Acrown). Twenty‐four‐h Acrown was modelled for shade‐tolerant Acer rubrum and shade‐intolerant Liriodendron tulipifera saplings growing for three years in a forest understorey under ambient and elevated [CO2] in free‐air CO2 enrichment.

2. Two factors best explained Acrown in ambient [CO2]: tree light environment and sapling allometry. Microsite light environment influenced carbon gain via daily photosynthetic photon flux (PFD), average diffuse PFD and sunfleck characteristics. Species differences in specific leaf area (SLA) and size‐related biomass allocation to leaves affected the effective leaf area and hence Acrown.

3. At a common above‐ground biomass, small saplings (100 g above‐ground dry mass) of L. tulipifera had higher Acrown than A. rubrum samples due to larger SLA and greater biomass allocation to leaves. Larger saplings of the two species had similar Acrown due to greater carbon allocation to leaves with increasing plant size in A. rubrum vs L. tulipifera. For saplings > 800 g, Acrown was greater in A. rubrum than in L. tulipifera. Enhancement of Acrown by elevated [CO2] on sunny days was similar for both species.

4. Overall, though the shade‐tolerant species had lower Acrown than the shade‐intolerant species at a common small size, our results indicate that the relative performance of these species can reverse at larger sizes due to allocational differences. These results suggest that elevated [CO2] may accelerate competition for light between A. rubrum and L. tulipifera as these species grow larger in the understorey.
Root system topology and diameter distribution of species from habitats differing in inundation frequency
Authors: Bouma T. J. , Nielsen K. L. , Van Hal J. , Koutstaal B.
Published: March 2003

We compared the root systems of seven halophytic species that occur at different elevations on a salt marsh, in order to (i) test the hypothesis that variations in root system architecture reflect adaptation to inundation frequency or nitrogen limitation, and (ii) verify the theoretically predicted relationships between root diameter, link magnitude and root topology. Diameters and lengths of individual laterals were determined along root axes, and branching patterns were quantified by calculating a topological index (TI).

 Chenopodiaceae (annual dicots) showed that with increasing elevation, the branch density and length of individual first‐order laterals tended to increase, so that the relative length of the main axes decreased. Root branching of the Chenopodiaceae at lower elevations was herringbone‐like, whereas species from higher elevations had smaller TIs because their branching patterns were more complex.

 The Gramineae, too, showed a tendency to increased length of individual laterals with increasing elevation. However, TI was not related to elevation, did not indicate a herringbone structure for all species, and was within the same range of that of the Chenopodiaceae.

 As root topology of the Chenopodiaceae is related to elevation, but that of the grasses is not, topology is not necessarily an important adaptive trait in all plant families that inhabit the salt marsh. Short first‐ and second‐order laterals may represent a more general architectural adaptation to frequent inundation, with longer first‐order laterals being favourable to competition for nutrients.

 Diameters at the root base tended to decrease if root branching was herringbone‐like (TI close to 1). Roots of first‐order laterals were approximately one‐third of the diameter of the main axes; second‐order laterals were approximately half the diameter of the first‐order laterals. These ratios illustrate the value of using the developmental segment‐ordering system in describing roots. The theoretically predicted relationship between root diameter and link magnitude was not present within individual orders of roots, whereas diameter did slowly increase with magnitude when combining different root orders.

 In the absence of a clear relationship between root diameter and link magnitude, the predicted high carbon costs associated with herringbone root systems disappear, whereas the advantage of minimized inter‐root competition remains. Consequently, herringbone root systems will be most efficient in terms of nutrients gained per carbon invested. However, dichotomous root systems offer a greater potential for exploring the soil, which contributes to the potential competitiveness of plants growing in nutrient limited habitats.
Root life spans of four grass species from habitats differing in nutrient availability
Authors: Van Der Krift T. A. J. , Berendse F.
Published: March 2003

In grass species that occur in pastures or hay meadows, life spans of roots determine much of the carbon and nutrient loss from the plant in addition to the amounts that are lost by mowing or grazing. We hypothesized that grass species from nutrient‐poor habitats had longer root life spans and consequently lost smaller quantities of nutrients through root turnover.

In a garden experiment, root life spans and root diameters were measured by repeated observations in minirhizotrons placed in monocultures of Lolium perenne L. and Arrhenatherum elatius L. (characteristic of fertile soils) and Molinia caerulea L. and Nardus stricta L. (preferring nutrient‐poor soils).

Average root life spans were 14 weeks in L. perenne, 40 weeks in A. elatius, 53 weeks in M. caerulea and 58 weeks in N. stricta. Root life spans of species from fertile habitats were significantly shorter than the root life spans of species from low fertility habitats.

In addition, there were significant differences in root diameter among species, root diameter being positively correlated to root life spans. Root diameter decreased during root ageing in all species, while the decline in diameter occurred more slowly in N. stricta than in A. elatius and M. caerulea.

 An increase in the abundance of plant species adapted to fertile habitats will, because of the greater C and nutrient returns to the soil in root turnover, increase soil fertility. This effect may constitute a positive feedback between changes in plant species composition and nutrient cycling.
Measuring drag without a force transducer: a terminal velocity assay
Authors: Loudon C. , Zhang J.
Published: March 2003

Organisms live surrounded by air or water, which exert drag on an organism when moving. These forces are significant ecologically because they can affect organisms’ distribution, behaviour or dispersal.

 Appropriate techniques for measuring or estimating these forces vary greatly depending on the magnitude of the forces and the flow pattern of the moving fluid (air or water; both gases and liquids are fluids). A simple method for estimating the drag in uniform steady flow is described. This technique is particularly well suited for forces of small magnitude (micronewtons) and slow flows (1 < Re < 100), and provides very uniform and well‐characterized ambient flow relative to the object.

  This method capitalizes on the force balance that exists when a falling object reaches terminal velocity. At terminal velocity, all of the forces sum to zero, and therefore the drag may be estimated from the other (known) forces (buoyancy and gravitational force). Orientation during falling may be controlled if necessary.
Repeatability estimates do not always set an upper limit to heritability
Authors: M. R. Dohm
Published: March 2003

The concept of repeatability, the measurement of consistent individual differences, has become an increasingly important tool in evolutionary and ecological physiology. Significant repeatability facilitates the study of selection acting on natural populations and the concept has several practical implications for identifying traits.

 When properly defined and measured, repeatability can set the upper limit to heritability. This is potentially a very useful interpretation of the repeatability of traits measured on natural populations because often, estimates of heritability cannot be obtained. Many recent reports of repeatability of individual differences for traits have made this interpretation.

 However, repeatability estimates may not set an upper limit to heritability if: (a) measured traits are not genetically identical, (b) common environmental effects work in opposition to direct genetic effects, (c) the temporary environments for each trait are negatively correlated, (d) significant genotype–environment interaction is present, or (e) the traits are influenced by maternal effects.

 The quantitative genetic theory that defines the concept of repeatability is reviewed and implications of violations of the five assumptions are discussed in the context of interpreting repeatability as an upper estimate to heritability.
High leaf mass per area of related species assemblages may reflect low rainfall and carbon isotope discrimination rather than low phosphorus and nitrogen concentrations
Authors: Lamont Byron B. , Groom Philip K. , Cowling R. M.
Published: March 2003

1. Leaf morphology at the site/species level should reflect environmental constraints on plant growth. One of the oldest controversies in ecology is the environmental basis for sclerophylly. The dominant view (Beadle’s theory) is that it has a nutritional, rather than a drought, basis, especially low phosphorus.

2. Using leaf mass per area (LMA) as an index of sclerophylly, we assessed its relationship with leaf phosphorus (P) and nitrogen (N) along extensive rainfall gradients in southwestern Australia and the Cape of South Africa. Leaf 13C/12C discrimination (Δ13C), as an index of intrinsic water‐use efficiency, was also examined in the Cape. All Hakea species (Proteaceae) were sampled at 10 sites in Australia (96 species), and all Proteaceae at 14 sites in the Cape (82 species). All were evergreen shrubs with iso(bi)‐lateral leaves.

3. In each region there was a strong (inverse) curvilinear relationship between mean LMA per site and mean annual rainfall and Δ13C, but none with mean P or N on a mass basis (although P and N on an area basis declined with rainfall). The Cape study was a particularly good test of Beadle’s theory, as P varied as much between sites as rainfall, and more between sites than within sites.

4. Leaf thickness and dry density were not as well correlated with rainfall as LMA, and leaf area and mass showed no relationship with rainfall. Area and mass had much greater variation within sites than between sites, limiting their value in plant–environment studies, while LMA was the most site‐stable of the eight leaf attributes measured, except for Δ13C.

5. For all species considered individually in each region, there was a similar pattern as the site level, with LMA most strongly correlated (negatively) with rainfall and Δ13C and (positively) with leaf thickness, but no consistent relationship with P, N or density.

6. We conclude that when water and nutrient supply vary independently in the field, rainfall (as an index of water status) and Δ13C may be more closely correlated (inversely) with level of sclerophylly than nutrient status among evergreens, so that the role of sclerophylly as a drought adaptation warrants further consideration.
Interspecific and spatial differences in nitrogen uptake in monocultures and two‐species mixtures in north European grasslands
Authors: Jumpponen A. , Högberg P. , Huss‐Danell K. , Mulder C. P. H.
Published: March 2003

1. To study the potential for complementarity in nitrogen acquisition from different soil depths, we injected an isotope tracer (15NH4Cl) at 5 and 20 cm depths in plant communities containing Achillea millefolium L. and Festuca ovina L. or Phleum pratense L. and Trifolium pratense L. in monocultures and two‐species mixtures.

2. In monoculture, Festuca and Phleum took up tracer at 5 and 20 cm depths. In contrast, Achillea and Trifolium monocultures acquired the tracer mainly from 5 cm depth. In two‐species mixtures, all four species took up tracer at 5 cm depth.

3. Achillea N acquisition from 20 cm depth increased in mixture with Festuca in comparison to that in monoculture; Festuca N acquisition from 20 cm depth decreased, although not significantly. Trifolium N acquisition remained unchanged when grown in mixture with Phleum. Phleum behaved like Festuca: its N acquisition from 20 cm depth in mixture was reduced in comparison to monoculture.

4. Our data on Festuca and Achillea support spatial partitioning in resource acquisition. This was not evident in Phleum and Trifolium mixture, potentially because Trifolium relied on N2 fixation as N source.

5. These results demonstrate spatial variation among plant species and plant communities in their N acquisition in the field.
Blood concentration of organochlorine pollutants and wing feather asymmetry in Glaucous Gulls
Authors: Bustnes J. O. , Folstad I. , Erikstad K. E. , Fjeld M. , Miland Ø. O. , Skaare J. U.
Published: March 2003

Non‐directional asymmetries (fluctuating asymmetry, FA) from bilateral symmetry in morphological traits have been used as an indicator of environmental stress and may become an important diagnostic tool in environmental sciences, such as ecotoxicology.

In this study the relationship was examined between wing feather asymmetry, measured as the difference between the length of the third primary on the left and right wing, and blood concentration of selected persistent organochlorines (OCs) in Arctic breeding Glaucous Gulls (Larus hyperboreus, Gunnerus).

There was a positive relationship between primary asymmetry and blood concentrations of two PCB (polychlorinated biphenyl) congeners (P < 0·05), oxychlordane (P < 0·05), DDE (p′p′‐dichlorodiphenyldichloroethylene) (P < 0·05), and especially HCB (hexachlorbenzene) (P < 0·001). At HCB levels above 30 ng g−1(wet mass) there was a 60% probability that the birds had asymmetric wing feathers.

This study indicated that the present levels of organochlorines in the European Arctic are stressors for Glaucous Gulls, and that asymmetry measurements of wing feathers may be used as an indicator of both exposure and effects of such contaminants. FA may also be a promising measurement for monitoring the early effects of organochlorine pollution on bird populations.
Effects of temperature and food quality on anuran larval growth and metamorphosis
Authors: Álvarez D. , Nicieza A. G.
Published: March 2003

Anurans exhibit high levels of growth‐mediated phenotypic plasticity in age and size at metamorphosis. Although temperature and food quality exert a strong influence on larval growth, little is known about the interacting effects of these factors on age and size at metamorphosis.

Plasticity in growth rates, maximum larval mass, mass loss, larval period and size at metamorphosis was examined in Iberian Painted Frogs (Discoglossus galganoi Capula, Nascetti, Lanza, Bullini & Crespo 1985) under different combinations of temperature and diet quality.

Temperature and diet had strong effects on the maximum size reached by tadpoles throughout the premetamorphic stages. Larval body mass varied inversely with temperature. The effect of diet depended on temperature; larvae fed on a ‘carnivorous’ diet (rich in protein and lipids) achieved a larger size than larvae offered an ‘herbivorous’ diet (rich in carbohydrates) at 17 °C but not at 12 or 22 °C.

Larval period was insensitive to diet composition, and varied only with temperature. Primarily the interacting effects of food quality and temperature affected size at metamorphosis. Size at metamorphosis varied inversely with temperature under the plant‐ and the animal‐based diets. However, the carnivorous diet resulted in bigger metamorphs at 17 and 22 °C, but did not influence final mass at 12 °C. Maximum size over the larval period explained most of the variation in mass loss after the premetamorphic growing phase.
Functional sites of human PCNA which interact with p21 (Cip1/Waf1), DNA polymerase δ and replication factor C
Authors: Oku Takashi , Ikeda Soichiro , Sasaki Hisashi , Fukuda Kotaro , Morioka Hiroshi , Ohtsuka Eiko , Yoshikawa Hiroshi , Tsurimoto Toshiki
Published: March 2003

Background:

PCNA, an eukaryotic DNA sliding clamp interacts with replication factors and the cell cycle protein, p21(Cip1/Waf1) and functions as a molecular switch for DNA elongation. To understand how DNA replication is regulated through PCNA, elucidation of the precise mechanisms of these protein interactions is necessary.

Results:

Loop‐region mutants in which human PCNA sequences were substituted with the corresponding Saccharomyces cerevisiae PCNA regions were prepared. Analysis of their functions, along with previously prepared alanine scanning mutants, demonstrated that some loops interact with DNA polymerase δ (pol δ) and replication factor C (RFC). The p21 binding sites of PCNA, mapped by affinity measurement of the mutant forms, found to be located within a distinct structure of the PCNA monomer, overlap with RFC‐ and pol δ‐interaction sites. Competition between p21 and pol δ or RFC for binding to PCNA results in efficient inhibition of its stimulation of pol δ DNA synthesis and RFC ATPase but not of PCNA loading on DNA by RFC.

Conclusions:

Semi‐saturated amounts of p21 selectively block formation of the active pol δ complex but not the RFC–PCNA complex at 3′‐ends of DNA primers. This differential effect may explain the specific inhibition of DNA replication by p21.
Escherichia coli HU protein suppresses DNA‐gyrase‐mediated illegitimate recombination and SOS induction
Authors: Shanado Yuka , Kato Jun‐ichi , Ikeda Hideo
Published: March 2003

Background

The HU protein is an abundant DNA binding protein of bacteria and is a major constituent of the bacterial nucleoid. HU protein is known to be involved in several fundamental biological functions, including DNA supercoiling, DNA replication, site‐specific DNA inversion, and transposition. It is generally thought that a functional relationship exists between HU protein and DNA gyrase.

Results

We found that an hupA hupB double mutant displays enhanced spontaneous illegitimate recombination during the formation of λbio transducing phage in Escherichia coli. Nucleotide sequence analysis of the resulting transducing phages showed that the E. coli bio and λ recombination sites did not have any homologous sequence. This mutation also enhanced the spontaneous expression of SOS functions. Furthermore, either overproduced GyrA protein or a temperature‐sensitive gyrB mutation suppressed the illegitimate recombination enhanced by the defect of HU protein.

Conclusion

These results show that the defect of HU induces illegitimate recombination and SOS response, which are probably mediated by DNA gyrase, implying that HU protein plays roles in suppression of illegitimate recombination and SOS response through interaction with DNA gyrase.
Role of p300, a transcriptional coactivator, in signalling of TGF‐β
Authors: Nishihara Ayako , Hanai Jun‐ichi , Okamoto Nobuaki , Yanagisawa Jun , Kato Shigeaki , Miyazono Kohei , Kawabata Masahiro
Published: March 2003

Background

Smad proteins are novel transcriptional regulators mediating the signalling of the transforming growth factor‐β (TGF‐β) superfamily. Coactivators such as p300/CBP promote transactivation by various transcription factors through a direct interaction with them. Adenoviral oncoprotein E1A, which binds p300, was shown to inhibit the signalling of TGF‐β. These findings raise the possibility that p300 may be involved in TGF‐β signalling.

Results

We investigated whether p300 is involved in transactivation by Smads. p300 enhanced the Smad‐induced transactivation of p3TP‐Lux, a TGF‐β responsive reporter. E1A inhibited this enhancement, and the inhibition required its ability to bind p300/CBP. p300 and Smad3, as well as Smad2, interacted in vivo in a ligand‐dependent manner. The binding region in Smad3 was its C‐terminal half that was previously shown to possess an intrinsic transactivation activity. The binding region in p300 was mapped to its C‐terminal 678 amino acids. The minimal Smad2/3‐interacting region, as well as the rest of the p300, inhibited the transactivation of p3TP‐Lux in a dominant‐negative fashion.

Conclusion

p300 interacted with Smad2 and Smad3 in a ligand‐dependent manner, and enhanced the transactivation by Smads. Our results present the molecular basis of the transactivation by Smad proteins.
S phase and differential DNA replication during Drosophila oogenesis
Authors: Royzman Irena , Orr‐Weaver Terry L.
Published: March 2003

A modified cell cycle, the endo cycle, produces the polyploid or polytene cells that are present in some tissues of most organisms. In the endo cycle, the S phase alternates with a gap phase, but mitosis does not occur. Genes needed to inhibit mitosis during the endo cycle and to promote the onset of S phase have been identified in Drosophila. Genomic intervals are differentially replicated during the endo cycle S phase such that some regions are under‐replicated, while others can be amplified. Cyclin E and E2F are needed for this differential DNA replication during Drosophila oogenesis.
Cooperation of two actin‐binding proteins, cofilin and Aip1, in Saccharomyces cerevisiae
Authors: Iida Kazuko , Yahara Ichiro
Published: March 2003

Background

Cofilin is a low‐molecular weight actin‐modulating protein, and is structurally and functionally conserved among eukaryotes. Cofilin is encoded by COF1 in Saccharomyces cerevisiae, and is essential for cell viability. Cofilin binds to and severs actin filaments in vitro, and also enhances their depolymerization. A partner protein that cooperates with cofilin in vivo has not been identified.

Results

When COF1 was over‐expressed in yeast cells under the GAL1 promoter in a medium containing galactose as a sole carbon source, the cells did not survive. These results indicate that cells can grow only when the expression of cofilin is appropriately regulated. Several temperature sensitive (ts‐) mutants were independently created by the random mutagenesis of COF1 with hydroxylamine. Mutated amino acids in ts‐mutants were mapped in the sequences that were presumed to be involved in actin binding. A gene on a multicopy plasmid which suppresses the ts‐phenotype of cof1‐101, a typical ts‐cofilin mutant, was isolated. The suppressor gene, SCF1, was found to be identical to AIP1, a gene encoding an actin‐interacting protein. Although SCF1/AIP1 is not essential for cell viability, a combination of cof1‐101 and Δscf1/aip1 is synthetic lethal. Immunofluorescence staining of a wild‐type strain using anti‐Aip1 antibodies revealed that Aip1 was distributed in cortical actin patches where cofilin was also co‐localized. Thick and long fibres stained with anti‐cofilin antibody were detected in Δscf1/aip1 cells, but not in SCF1/AIP1 cells.

Conclusions

These results suggest the cooperative modulation of the actin cytoskeleton by cofilin and Aip1.
DCRY is a Drosophila photoreceptor protein implicated in light entrainment of circadian rhythm
Authors: Ishikawa Tomoko , Matsumoto Akira , Kato Jr Tomohisa , Togashi Shin , Ryo Haruko , Ikenaga Mituo , Todo Takeshi , Ueda Ryu , Tanimura Teiichi
Published: March 2003

Background

: Light is the major environmental signal for the entrainment of circadian rhythms. In Drosophila melanogaster, the period(per) and timeless (tim) genes are required for circadian behavioural rhythms and their expression levels undergo circadian fluctuations. Light signals can entrain these rhythms by shifting their phases. However, little is known about the molecular mechanism for the perception and transduction of the light signal. The members of the photolyase/cryptochrome family contain flavin adenine dinucleotide (FAD) as chromophore and are involved in two diverse functions, DNA repair and photoreception of environmental light signals.

Results

: We report the cloning of a new member of this family, dcry, from Drosophila. Northern blot analysis shows that this gene is expressed in various tissues. The dcry mRNA is expressed in a circadian manner in adult heads, while such rhythmic fluctuation is abolished in the clock‐defective per0 and tim0 mutants. The circadian expression is dampened down in constant darkness. The over‐expression of the dcry gene alters the light‐induced phase delay in the locomotor activity rhythms of flies.

Conclusion

: These results suggest that DCRY is a circadian photoreceptor and that its expression is regulated by circadian clock genes.
Severe osteopetrosis, defective interleukin‐1 signalling and lymph node organogenesis in TRAF6‐deficient mice
Authors: Naito Asuka , Azuma Sakura , Tanaka Sakae , Miyazaki Tsuyoshi , Takaki Satoshi , Takatsu Kiyoshi , Nakao Kazuki , Nakamura Kenji , Katsuki Motoya , Yamamoto Tadashi , Inoue Jun‐ichiro
Published: March 2003

Background

TRAF6, a member of the tumour necrosis factor receptor‐associated factor family, was first identified as a transducer of CD40 and interleukin‐1 receptor (IL‐1R) signals based on the interaction of TRAF6 with the cytoplasmic tail of CD40 and with the IL‐1R associated kinase in vitro. However, the functions of TRAF6 in vivo remain unidentified.

Results

We show that TRAF6−/− mice exhibit severe osteopetrosis and are defective in osteoclast formation. In vitro culture experiments revealed that osteoclast precursor cells derived from TRAF6−/− mice are unable to differentiate to functional osteoclasts in response to osteoclast differentiation factor (ODF). In bone marrow of TRAF6−/− mice, the number of sIgM+B220+ immature B cells is markedly reduced while the ratio of proB to preB cells is not affected. In contrast, development of thymocytes is not affected. Furthermore, TRAF6−/− mice are defective in lymph node organogenesis and IL‐1 signalling in thymocytes.

Conclusions

The results identify TRAF6 as an essential component of ODF signalling pathway, and also show that TRAF6 plays pivotal roles in immune and inflammatory systems in vivo.
Two separate sequences of PB2 subunit constitute the RNA cap‐binding site of influenza virus RNA polymerase
Authors: Honda Ayae , Mizumoto Kiyohiso , Ishihama Akira
Published: March 2003

Background

Influenza virus RNA polymerase with the subunit composition of PB1‐PB2‐PA is a unique multifunctional enzyme with the activities of both synthesis and cleavage of RNA, and is involved in both transcription and replication of the RNA genome. Transcription is initiated by using capped RNA fragments, which are generated after cleavage of host cell mRNA by the RNA polymerase‐associated capped RNA endonuclease. To identify the RNA cap 1‐binding site on the RNA polymerase, viral ribonucleoprotein (RNP) cores were subjected to UV‐crosslinking with RNA which was labelled with 32P only at the cap‐1 structure.

Results

After SDS‐PAGE of UV‐crosslinked cores, 32P was found to be associated only with the PB2 subunit (759 amino acid residues). The labelled PB2 was subjected, together with PB2 expressed in E. coli, to limited digestion with V8 protease. Analysis of the amino terminal sequences of some isolated fragments with the crosslinked cap‐1 indicated that two separate sequences within the PB2 were involved in RNA cap‐1 binding, one (N‐site) at the N‐terminal proximal region approximately between amino acid residues 242–282 downstream from the PB1 subunit‐binding site and the other (C‐site) between residues 538–577 including the cap‐binding motifs. Two lines of evidence support the prediction of the involvement of two separate PB2 sequences on the RNA cap‐binding: (i) cross‐linking of the capped RNA on to expressed and isolated PB2 fragments, each containing either the N‐site or the C‐site; and (ii) competition of capped RNA‐binding to PB2 by both of the N‐ and C‐terminal PB2 fragments. Taking together, we propose that two separate sequences within PB2 constitute the capped RNA‐binding site of the RNA polymerase.

Conclusion

Two separate sequences, one N‐(242–282) and the other C‐terminal (538–577) proximal segments of PB2 subunit, constitute the RNA cap‐binding site of the influenza virus RNA polymerase.
A Hrs binding protein having a Src homology 3 domain is involved in intracellular degradation of growth factors and their receptors
Authors: Takata Hiroyuki , Kato Masaki , Denda Kimitoshi , Kitamura Naomi
Published: March 2003

Background

Hrs (hepatocyte growth factor (HGF)‐regulated tyrosine kinase substrate) is an early endosomal protein that is rapidly tyrosine‐phosphorylated in cells stimulated with growth factors. Hrs is thought to play a regulatory role in the endocytosis of growth factor/receptor complexes through early endosomes. In this study, we searched for Hrs‐interacting molecules which may regulate the function of Hrs, using a yeast two‐hybrid system.

Results

We isolated a cDNA clone encoding a novel Src homology 3 (SH3)‐containing protein, and named it ‘Hrs binding protein’ (Hbp). Hbp was co‐immunoprecipitated with Hrs, and its intracellular localization was similar to that of Hrs. The association between Hbp and Hrs was mediated through the coiled coil motifs in Hbp and Hrs. Deletion mutants of Hbp lacking either the SH3 domain or the Hrs binding domain showed dominantly negative effects on the intracellular degradation of a growth factor and its receptor, but not on the internalization of growth factor/receptor complexes.

Conclusions

Hbp is thought to be closely associated with Hrs on early endosomes. Hbp, together with Hrs may play a regulatory role in the vesicular transport of growth factor/receptor complexes through early endosomes, for their degradation.
NF‐κB activation through IKK‐i‐dependent I‐TRAF/TANK phosphorylation
Authors: Nomura  , Kawai  , Nakanishi Kenji , Akira Shizuo
Published: March 2003

Background

NF‐κB is an ubiquitously expressed transcription factor that plays an important role in the immune, anti‐apoptotic and inflammatory responses. NF‐κB is normally sequestered in the cytoplasm by interacting with inhibitory IκB molecules. Upon stimulation, IκB is phosphorylated and subsequently degraded by the proteasome, allowing NF‐κB to translocate into the nucleus where they regulate target gene expression. Two kinases, IKK‐α and IKK‐β, which are responsible for IκB phosphorylation were recently identified. We have recently identified a cytokine inducible IKK‐i, a kinase related to IKK‐α and ‐β. IKK‐i significantly induced NF‐κB activation upon over‐expression, as did IKK‐α and IKK‐β. Unlike IKK‐α and IKK‐β, IKK‐i phosphorylated Ser36 but not Ser32 in vitro, suggesting that IKK‐i activates NF‐κB by distinct mechanisms from the conventional IKKs.

Results

I‐TRAF/TANK was isolated as a molecule that interacts specifically with inducible IκB kinase (IKK‐i) by the yeast two‐hybrid screening procedure. The association of IKK‐i and I‐TRAF is mediated via the interaction between the N‐terminal domain of I‐TRAF and the C‐terminal portion of IKK‐i. In vitro kinase assays demonstrate that IKK‐i phosphorylates I‐TRAF in the middle portion that associates with TRAF2. Interestingly, TRAF2 is freed from the I‐TRAF/TRAF2 complex after I‐TRAF phosphorylation. NF‐κB activation by IKK‐i is significantly blocked by coexpression of the N‐terminal domain of I‐TRAF, dominant negative TRAF2, and dominant negative NIK and IKK‐β. IKK‐i over‐expression also induced c‐Jun N‐terminal kinase. These results show that I‐TRAF is a substrate of IKK‐i. NF‐κB activation by IKK‐i may be mediated through phosphorylation of I‐TRAF by IKK‐i and subsequent liberation of TRAF2.

Conclusion

These results indicate that NF‐κB activation by IKK‐i is mediated through phosphorylation of I‐TRAF/TANK by IKK‐i and subsequent liberation of TRAF2.
Identification and characterization of testis specific ornithine decarboxylase antizyme (OAZ‐t) gene: expression in haploid germ cells and polyamine‐induced frameshifting
Authors: Tosaka Yasuhiro , Tanaka Hiromitsu , Yano Yoshihisa , Masai Kumiko , Nozaki Masami , Yomogida Kentaro , Otani Shuzo , Nojima Hiroshi , Nishimune Yoshitake
Published: March 2003

Background

Polyamines are known to play important roles in the proliferation and differentiation of many types of cells. However, in the testis, where polyamines such as spermidine and spermine exist in high concentrations, their roles still remains to be elucidated.

Results

We have cloned a testis‐specific gene encoding an ornithine decarboxylase antizyme known to control intracellular concentrations of polyamines in a feedback manner. The mRNA encoding the protein named ornithine decarboxylase antizyme in testis (OAZ‐t) was specifically expressed in haploid germ cells. In contrast, the mRNA level of somatic ornithine decarboxylase antizyme 1 (OAZ1) decreased markedly at the late stages of haploid germ cell differentiation. OAZ‐t mRNA was first observed in 23‐day‐old mice, whereas the OAZ‐t protein was detected much later, at 35 days after birth. Further experiments on OAZ‐t revealed that polyamines were capable of inducing a frameshifting at the frameshift sequence of OAZ‐t mRNA, resulting in the translation of OAZ‐t, as was the case with the somatic OAZ1. Transfection of OAZ‐t cDNA inactivated the ornithine decarboxylase activity in the HEK293 cells.

Conclusions

Results indicate that the expression of OAZ‐t is controlled at both transcriptional and translational levels, and that OAZ‐t likely plays a key role in spermatogenesis by regulating the intracellular concentration of polyamines in haploid germ cells.
MST1‐JNK promotes apoptosis via caspase‐dependent and independent pathways
Authors: Ura Seiji , Masuyama Norihisa , Graves Jonathan D. , Gotoh Yukiko
Published: March 2003

Background MST1 is an upstream kinase of the JNK and p38 MAPK pathways whose expression induces apoptotic morphological changes such as nuclear condensation. During apoptosis, caspase cleavage of MST1 removes a C‐terminal regulatory domain, increasing the kinase activity of the MST1 N‐terminal domain. Downstream pathways of MST1 in the induction of apoptosis remain to be clarified.

Results
In this study, we found that the expression of MST1 resulted in caspase‐3 activation. Therefore, MST1 is not only a target of caspases but also an activator of caspases. This caspase activation and apoptotic changes occur through JNK, since the co‐expression of a dominant‐negative mutant of JNK inhibited MST1‐induced morphological changes as well as caspase activation. In contrast, neither a dominant‐negative p38 nor the p38 inhibitor SB203580 inhibited them. MST1 induced nucleosomal DNA fragmentation, which was suppressed by caspase inhibitors or ICAD (Inhibitor of Caspase‐Activated DNase). Surprisingly, however, other changes such as membrane blebbing and chromatin condensation were not inhibited by caspase inhibitors.

Conclusion
These results suggest that MST1 most likely promotes two events through JNK activation; first, MST1 induces the activation of caspases, resulting in CAD‐mediated DNA fragmentation, and second, MST1 induces chromatin condensation and membrane blebbing without utilizing downstream caspases.
Bacterial SsrA system plays a role in coping with unwanted translational readthrough caused by suppressor tRNAs
Authors: Ueda Koji , Yamamoto Yasufumi , Ogawa Kazuko , Abo Tatsuhiko , Inokuchi Hachiro , Aiba Hiroji
Published: March 2003

Backgrounds: Bacterial SsrA RNA (also known as tmRNA or 10Sa RNA) mediates the addition of a short peptide tag to the C‐terminus of the nascent polypeptide when a ribosome is stalled at the 3′ end of an mRNA lacking a stop codon. This process, called trans‐translation, rescues the stalled ribosome and ensures degradation of tagged polypeptides by ATP‐dependent proteases. To fully understand the physiological roles of SsrA RNA, it is essential to know how endogenous mRNA targets for the SsrA system are generated in cells. The aim of the present study is to examine how translational readthrough by suppressor tRNAs affects trans‐translation in Escherichia coli.

Results: We demonstrated that SsrA tagging of bulk cellular proteins was significantly enhanced by an ochre or an amber suppressor tRNA. Western blot analysis of proteins produced from specific genes possessing a Rho‐independent terminator revealed that readthrough at the normal stop codon leads to an efficient tagging and proteolysis of the extended proteins. Size analyses of both protein and mRNA suggested that tagging of extended proteins occurs because ribosome passing through the normal stop codon presumably reach the 3′ end of mRNA defined by the transcription terminator hairpin. The inhibitory effect of ssrA mutation on cell growth was markedly amplified in cells with an ochre suppressor tRNA.

Conclusion: The present finding suggests that the SsrA system contributes to scavenge errors and/or problems caused by translational readthrough that occurs typically in the presence of a suppressor tRNA.
The CLN3/SWI6/CLN2 pathway and SNF1 act sequentially to regulate meiotic initiation in Saccharomyces cerevisiae
Authors: Purnapatre Kedar , Piccirillo Sarah , Schneider Brandt L. , Honigberg Saul M.
Published: March 2003

Background: IME1, which is required for the initiation of meiosis, is regulated by Cln3:Cdc28 kinase, which activates the G1‐to‐S transition, and Snf1 kinase, which mediates glucose repression. Here we examine the pathway by which Cln3:Cdc28p represses IME1 and the relationship between Cln3:Cdc28p and Snf1p in this regulation.

Results: When wild‐type yeast cease growth, they express IME1 to moderate levels, intermediate between the low levels expressed during growth and the high levels expressed during sporulation. Moderate IME1 expression occurred in cln3Δ, cln1Δ cln2Δ, cdc28‐4 and swi6Δ mutants, even during growth. These mutants also induced IME1 expression more rapidly than the wild‐type. CLN3 required SWI6 and CLN2 to repress IME1 and IME2, but CLN1 was much less active than CLN2 in this repression. The phenotype of the cln3Δ snf1Δ double mutant indicated that Cln3:Cdc28p regulates IME1 independently of SNF1.

Conclusion: Entry into meiosis involves two independent but sequential controls, which regulate IME1 via a three position switch: (i) during growth IME1 is repressed by the CLN3/SWI6/CLN2 pathway, (ii) once growth ceases, this repression is released and IME1 is expressed at moderate levels, and (iii) subsequently, nutritional conditions that activate Snf1p allow high IME1 expression.
A role of the C‐terminus of aquaporin 4 in its membrane expression in cultured astrocytes
Authors: Nakahama Ken‐ichi , Fujioka Atsuko , Nagano Mamoru , Satoh Shinsuke , Furukawa Kei‐ichi , Sasaki Hiroshi , Shigeyoshi Yasufumi
Published: March 2003

Background: Aquaporin 4 (AQP4) is a predominant water channel protein in mammalian brains, which is localized in the astrocyte plasma membrane. Membrane targeting of AQP4 is essential to perform its function. The mechanism(s) of membrane targeting is not clear in astrocytes.

Results: We investigated the role of the C‐terminus of AQP4 (short isoform) in its membrane targeting by an expression study of C‐terminal mutants of AQP4 in cultured astrocytes. The deletion of 26 C‐terminal residues of AQP4 (AQP4Δ276–301aa) results in the intracellular localization of the protein. However, smaller deletions than 21 C‐terminal residues did not alter its plasma membrane localization. These results suggest that C‐terminal residues between Val276 and Ile280 play an important role in the expression of AQP4 in the plasma membrane. However, the plasma membrane localization of the AQP4(A276AAAA280) mutant (alanine substitution of Val276‐Ile280 of AQP4) suggests that another signal for membrane targeting exists in the C‐terminus of AQP4. The deletion or point mutations of the PDZ binding motif of the AQP4(A276AAAA280) mutant resulted in the intracellular localization of the proteins. These results suggest that the PDZ binding motif may also be involved in the membrane targeting of AQP4.

Conclusions: We found that the C‐terminal sequence of AQP4 contains two important signals for membrane expression of AQP4 in cultured astrocytes. One is a hydrophobic domain and the other is a PDZ binding motif that exists in the C‐terminus.
Identification of the interactive interface and phylogenic conservation of the Nrf2‐Keap1 system
Authors: Kobayashi Makoto , Itoh Ken , Suzuki Takafumi , Osanai Hitoshi , Nishikawa Keizo , Katoh Yasutake , Takagi Yaeko , Yamamoto Masayuki
Published: March 2003

Background:  The transcription factor Nrf2 and its negative regulator Keap1 play important roles in transcriptional induction of a set of detoxifying and anti‐oxidant enzymes. To gain an insight into our present enigma as to how cells receive oxidative and electrophilic signals and transduce them to Nrf2, we have developed a zebrafish model system for molecular toxicological studies.

Results: We systematically cloned zebrafish cytoprotective enzyme cDNAs and found their expression to be efficiently induced by electrophilic agents. We consequently identified the presence of Nrf2 and Keap1 in zebrafish. Both loss‐ and gain‐of‐function analyses demonstrated that Nrf2 is the primary regulator of a subset of cytoprotective enzyme genes, while Keap1 suppresses Nrf2 activity in zebrafish. An ETGE motif, critical for the Nrf2–Keap1 interaction, was identified in the Neh2 domain of Nrf2 by reverse two‐hybrid screening and found to be indispensable for the regulation of Nrf2 activity in zebrafish.

Conclusion: Taken together, these results indicate that the Nrf2‐Keap1 system is highly conserved among vertebrates and that the interface between Nrf2 and Keap1 forms an important molecular basis of this regulatory system.
PCNA clamp facilitates action of DNA cytosine methyltransferase 1 on hemimethylated DNA
Authors: Iida Tetsuo , Suetake Isao , Tajima Shoji , Morioka Hiroshi , Ohta Satoshi , Obuse Chikashi , Tsurimoto Toshiki
Published: March 2003

Background: Proliferating cell nuclear antigen (PCNA) is a ring‐shaped protein known as a processivity factor of DNA polymerase δ. In addition to this role, PCNA interacts with a number of other proteins to increase their local concentration at replicated DNA sites. DNA cytosine methyltransferase 1 (Dnmt1), which preserves epigenetic signals by completing the methylation of hemimethylated DNA after DNA replication, has been indicated as one of these PCNA binding proteins by a previous work. However, the molecular mechanisms and functional significance of their association have not yet been studied.

Results: Dnmt1 can be readily isolated from nuclear extracts by PCNA affinity chromatography. Studies of the interactions between the two proteins demonstrate that the N‐terminal region of Dnmt1, which contains a typical PCNA binding motif, has core PCNA binding activity, and that the remaining portion of the protein exerts a negative influence on the interaction of Dnmt1 with PCNA. The affinity of Dnmt1 for DNA is much higher for DNA bound by PCNA than for free DNA. Furthermore, DNA methylation assays with hemimethylated DNA as a substrate revealed that PCNA clamp‐bound DNA is methylated more efficiently by Dnmt1 than is free DNA.

Conclusion: These results provide the first biochemical evidence that physical interactions between PCNA and Dnmt1 facilitate the methylation of newly neplicated DNA, on which PCNA remains associated as a functional clamp.
Fossils in folklore
Authors: TAYLOR PAUL D.
Published: March 2003

Fossils have been known to people since at least Palaeolithic times, when they were employed decoratively. However, it was not until the 17th century that the true origin of fossils became widely acknowledged. In the interim period, a rich folklore developed around fossils, including some bizarre beliefs about their origins and medicinal and magical properties. Names such as thunderbolts, tonguestones, toadstones, snakestones and Devil's Toenails became widely used for different types of fossils in Britain. Fossil folklore not only played an important part in the early history of palaeontology but also serves to underline the role of comparative morphology and the need for living analogues if we are to interpret fossils correctly.
Palaeozoic corals: their evolution and palaeoecology
Authors: Scrutton Colin
Published: March 2003

In recent years, our understanding of Palaeozoic corals has increased enormously. Several new groups have been discovered, extending the range of corals back to the early Cambrian, and we now have a clearer idea of the relationships between them. None of them was a direct ancestor to the post‐Palaeozoic corals. Similarly, the ecology of Palaeozoic corals differed from that of their living relatives in important respects. Palaeozoic corals were principally adapted for life on soft substrates and were not the spectacular reef‐builders that modern corals have become. However, their contribution to Palaeozoic ecosystems is nonetheless fascinating. Their structural adaptations and growth‐forms can tell us much about life and processes on Palaeozoic sea floors.