bluuebunny/crossref_metadata_2025 huggingface dataset:
The significance of differences in the mechanisms of photosynthetic acclimation to light, nitrogen and CO2 for return on investment in leaves
Authors: Sims D. A. , Seemann J. R. , Luo Y.
Published: March 2003

1. We report changes in photosynthetic capacity of leaves developed in varying photon flux density (PFD), nitrogen supply and CO2 concentration. We determined the relative effect of these environmental factors on photosynthetic capacity per unit leaf volume as well as the volume of tissue per unit leaf area. We calculated resource‐use efficiencies from the photosynthetic capacities and measurements of leaf dry mass, carbohydrates and nitrogen content.

2. There were clear differences between the mechanisms of photosynthetic acclimation to PFD, nitrogen supply and CO2. PFD primarily affected volume of tissue per unit area whereas nitrogen supply primarily affected photosynthetic capacity per unit volume. CO2 concentration affected both of these parameters and interacted strongly with the PFD and nitrogen treatments.

3. Photosynthetic capacity per unit carbon invested in leaves increased in the low PFD, high nitrogen and low CO2 treatments. Photosynthetic capacity per unit nitrogen was significantly affected only by nitrogen supply.

4. The responses to low PFD and low nitrogen appear to function to increase the efficiency of utilization of the limiting resource. However, the responses to elevated CO2 in the high PFD and high nitrogen treatments suggest that high CO2 can result in a situation where growth is not limited by either carbon or nitrogen supply. Limitation of growth at elevated CO2 appears to result from internal plant factors that limit utilization of carbohydrates at sinks and/or transport of carbohydrates to sinks.
Responses to elevated temperature and CO2 in the perennial grass Agrostis curtisii in relation to population origin
Authors: Norton L. R. , Firbank L. G. , Gray A. J. , Watkinson A. R.
Published: March 2003

1. Evolutionary responses to climate change will depend on the presence of heritable variation within species populations for traits that increase fitness under the changing conditions. Patterns of ecotypic differentiation in relation to latitude in some species suggest that such variation exists in relation to temperature responses. Response to elevated CO2, whether heritable or not, is not expected to be related to latitudinal or climatic differences within temperate regions.

2. To test these ideas, seeds were collected from 10 populations of the outbreeding perennial grass Agrostis curtisii across its range in Europe from south Wales to Portugal. Plants were grown under ambient and elevated temperature and CO2 conditions, in a factorial design, in solardomes; two half sibs from each population were planted in separate pots in each of the two replicate domes with each combination of treatments. One half sib was harvested at the end of the first summer, the second at the end of the second summer.

3. Survival was uniformly high and flowering uniformly low across treatments and populations.

4. Responses to temperature and CO2 treatments varied over time for almost all populations. Treatment effects were not significant on plants harvested in year 1, although there was a trend towards higher shoot biomass under the elevated temperature and CO2 treatment. In year 2 shoot biomass was significantly higher under the elevated temperature treatment across all populations and there was a strong trend towards decreased biomass under elevated CO2.

5. There were no significant correlations of plant response to either CO2 or temperature with climate at origin.

6. These results warn of the dangers of extrapolating evolutionary plant responses to CO2 from short‐term experiments.
Aerobic performance does not affect social rank in female Red Jungle Fowl
Authors: CHAPPELL M. A. , ZUK M. , JOHNSEN T. S.
Published: March 2003

1. Exercise capacity ultimately constrains behaviour, and therefore may influence social interactions. The hypothesis was tested that individual differences in maximal rates of oxygen consumption (V·O2max) – a primary determinant of sustainable exercise capacity – affect dominance hierarchies in experimental all‐female flocks of the highly social Red Jungle Fowl (Gallus gallus). It was also examined whether social rank could in turn influence V·O2max (which is quite plastic in most vertebrates), and whether these relationships were influenced by a common and relatively benign parasite, the intestinal nematode Ascaridia galli.

2. Substantial between‐individual variation was found in V·O2max that was significantly repeatable over time, but there was no indication that V·O2max was affected by A. galli infection. Stable social hierarchies were quickly established in 26 of 28 experimental flocks (each contained three females previously isolated from each other). Infection status affected social rank, but there was no consistent pattern between rank and infection.

3. No indication was found that individual differences in V·O2max either predicted the social rank in newly formed flocks, or were affected by social status in established flocks.
Seedling survival of Mediterranean shrubland species in relation to root:shoot ratio, seed size and water and nitrogen use
Authors: LLORET F. , CASANOVAS C. , PEÑUELAS J.
Published: March 2003

1. We hypothesized that in Mediterranean plant communities seedling survival of different species during the drought period would be related to their ability to use below‐ground resources, particularly water and nitrogen.

2. For 5 years we studied under field conditions the summer seedling survival of 11 dominant species of a Spanish Mediterranean shrubland. We related seedling survival of the different species to their seed size and root allocation estimated as the slope of the function root biomass vs log shoot biomass. We used δ13C for the estimation of water‐use efficiency and δ15N and nitrogen concentration to determine the sources of nitrogen utilized. We correlated these variables with root allocation.

3. Seedling survival of the different species was positively correlated with root allocation and seed size. Root allocation was also positively correlated with seed size. δ15N and nitrogen concentration were also positively correlated with root allocation, but δ13C was not.

4. Under the relatively moist conditions occurring during summer 1996, higher root allocation was associated with the use of nitrogen from more nitrogen saturated microsites.
Photosynthesis and stomatal conductance of mature canopy Oak (Quercus robur) and Sycamore (Acer pseudoplatanus) trees throughout the growing season
Authors: Morecroft M. D. , Roberts J. M.
Published: March 2003

1. Gas exchange is strongly seasonal in temperate deciduous forests; however, virtually no studies have described measurements made at the leaf scale through the whole growing season. This level of detail is important to understanding processes as substantial differences may occur within the canopy between species and between sun and shade leaves.

2. Gas exchange was measured weekly throughout the growing season in leaves of Oak (Quercus robur) and Sycamore (Acer pseudoplatanus) trees at Wytham Woods, UK. In Q. robur sun, shade and intermediate leaves were sampled on each occasion.

3.
Acer pseudoplatanus came into leaf before Q. robur and commenced net assimilation (A) earlier. Maximum irradiance saturated photosynthetic rates (Amax) in Q. robur were reached approximately 50 days after budburst and were maintained until approximately 20 days before abscission. Acer pseudoplatanus lost its leaves before Q. robur in the autumn and showed no seasonal trend in Amax. The lag between budburst and attainment of maximum photosynthetic rate in Q. robur was substantial and represented a quarter of the potential growing season.

4.
A
max of A. pseudoplatanus (3·5 μmol m–2 s–1) sun leaves was substantially lower than that of Q. robur (10·4 μmol m–2 s–1) despite A. pseudoplatanus being a fast‐growing species. The earlier flushing of A. pseudoplatanus leaves in the spring could not completely compensate for this. It is likely that other variables, such as total leaf area, are responsible for the high growth rate of A. pseudoplatanus, rather than high photosynthetic rates.

5. Shade leaves of Q. robur had lower Amax and mean A than sun leaves; their quantum efficiency was also lower. Consistent with this, sun leaves had a higher nitrogen content per unit area, resulting from a lower specific leaf area (SLA).

6. Week to week variations in solar radiation influenced A; this could be described by a non‐rectangular hyperbola. Stomatal conductance (gs) decreased in conditions of high vapour pressure deficit (VPD). However, this did not decrease A as high VPD was associated with high solar radiation and the stimulation of photosynthesis caused by high photosynthetic photon flux density (PPFD) more than compensated for the reduction by VPD.
The functional relationship between peak spring floods and survival and growth of juvenile Atlantic Salmon (Salmo salar) and Brown Trout (Salmo trutta)
Authors: Jensen A. J. , Johnsen B. O.
Published: March 2003

1.The effects of high spring floods on survival and growth of juvenile Atlantic Salmon,Salmo salar,and Brown Trout,Salmo trutta, are explored, using data from a long‐term study in the River Saltdalselv, northern Norway. The flow regime in this river is typical for northern rivers.

2.There was considerable variation in year class strength of both species.

3.Mortality of Atlantic Salmon increased significantly in years with high discharge during the alevin stage as well as the first week after emergence. High discharge during the egg stage and more than 1 week after emergence seemed to be of minor importance. Water temperature at emergence was rather high (average 10·5 °C) and did not significantly affect year class strength.

4.Brown Trout emerged earlier than Atlantic Salmon at an average water temperature of 8·2 °C. Highest mortality was observed in years with low water temperatures at emergence as well as high discharge during the alevin stage.

5.For 1‐year‐old fish or older, the size of the spring peak flood did not influence mortality significantly.

6.Growth of Atlantic Salmon parr was diminished in years with a high peak spring flood. A similar effect on Brown Trout was not detected.
Influence of leaf chemistry of Lotus corniculatus (Fabaceae) on larval development of Polyommatus icarus (Lepidoptera, Lycaenidae): effects of elevated CO2 and plant genotype
Authors: Goverde M. , Bazin A. , Shykoff J. A. , Erhardt A.
Published: March 2003

1. Four Lotus corniculatus genotypes differing in cyanoglycoside and condensed tannin concentrations were grown in either low (350 ppm) or high (700 ppm) atmospheric CO2 environments. Larval performance, consumption and conversion efficiency of Polyommatus icarus feeding on this plant material were measured.

2. Plants grown under elevated CO2 contained less cyanoglycosides, more condensed tannins and more starch than control plants. However, water concentration, nitrogen and protein as well as nitrogen concentration in relation to carbon concentration did not differ between CO2 treatments.

3. The four genotypes differed significantly in condensed tannins, cyanoglucoside, leaf water and leaf nitrogen but no genotype–CO2 interaction was detected, except for total phenolics and condensed tannins in which two plant genotypes showed stronger increases under elevated CO2 than the other two.

4. Larvae of P. icarus consumed more plant material and used and converted it more efficiently from plants grown at high atmospheric CO2.

5. Larvae developed significantly faster and were significantly heavier when fed plant material grown under elevated CO2. The observed difference in mass disappeared in the pupal and adult stages. However, lipid concentration of adults from the elevated CO2 treatment was marginally significantly higher than of controls.

6. It is concluded that the higher carbohydrate concentration of L. corniculatus plants grown at elevated CO2 renders leaves more suitable and better digestible to P. icarus. Furthermore, differences in allelochemicals might influence the palatability of L. corniculatus leaves for this specialist on Fabaceae.
Partitioning variance in a physiological trait: desiccation resistance in keratin beetles (Coleoptera, Trogidae)
Authors: Chown S. L. , Le Lagadec M. D. , Scholtz C. H.
Published: March 2003

1.Although variation in physiological traits forms the substance of evolutionary physiology, the way that this variation is partitioned among different hierarchical levels (e.g. population, species, genus) is not well known.

2.In this study variance partitioning is examined in body size, water content, lipid content, rate of water loss, maximum tolerable water loss and survival time at four levels (individual, population, species, genus) in southern African keratin beetles.

3.It is found that most variance in body size, and the physiological traits that are strongly influenced by body size (water and lipid content, maximum tolerable water loss, rate of water loss), is partitioned at the generic level (50–70%), then at the species level (20–50%) and finally at the population (1–9%) and individual (2–18%) levels.

4.On the other hand, variance in survival time, and variance in rate of maximum water loss once the effects of body size have been taken into account, are partitioned mostly at the species level (40–70%), whereas maximum tolerable water loss and lipid and water content show greatest variance at the individual level (63–75%). This is largely a consequence of differences in the extent of scaling of these traits.

5.The results suggest that where possible the effects of phylogeny should be controlled for when examining desiccation resistance in insects using comparative methods, but that confidence in conclusions from previous studies, which have used mass‐specific data, is not unwarranted.
Flight muscle atrophy and predation risk in breeding birds
Authors: Veasey J. S. , Houston D. C. , Metcalfe N. B.
Published: March 2003

1. The speed with which small birds can get airborne is critical to the effectiveness of their escape response when attacked by a predator. However, take‐off ability is likely to be affected by physiological changes occurring as a result of egg formation.

2. To investigate whether reduced take‐off velocity is a cost of reproduction, the physiological costs of egg production in the Zebra Finch (Taeniopygia guttata) were experimentally manipulated by varying both the number of eggs a female laid and the quality of her prelaying diet. The effect of changes in postlaying flight muscle condition and body mass upon alarmed flight take‐off velocity (a measure of escape‐ability in birds) was subsequently measured.

3. Changes in muscle condition were found to correlate positively with changes in various measures of flight velocity: treatments that caused the greatest declines in muscle condition during egg‐laying were associated with the greatest declines in flight performance over this period. In contrast, breeding attempts that caused the smallest declines in muscle condition were associated with improvements in flight performance (i.e. birds flew faster at the end of laying than at the start).

4. These effects were independent of changes in body mass, and occurred postlaying, suggesting that the cost of egg production lies primarily in the formation of the eggs, rather than in carrying them as other studies had suggested. The observed trade‐off between muscle loss resulting from egg production and escape ability could have important implications for the evolution of optimal clutch size in birds.
Biological diversity and probability of local extinction of ecosystems
Authors: Nijs I. , Impens I.
Published: March 2003

1. With a mathematical model, we demonstrate that two principle components of biological diversity have antagonistic effects on the probability of ecosystem functioning (i.e. on ecosystem reliability, sensuNaeem 1998). Increasing the number of functionally analogous species in the system is shown to enhance reliability, while increasing the number of functions or processes which these species support (functional diversity) has a negative influence.

2. Probability of functioning was calculated from the likelihood that essential ecosystem functions are lost, which was based on the probability of local extinction of the species that support these functions.

3. A third component of diversity, heterogeneity among extinction probabilities of different species, enhanced probability of ecosystem functioning for species belonging to the same functional group, but only in species‐poor systems. In contrast, heterogeneity among species of different functional groups drastically reduced probability of ecosystem functioning and this reduction was more pronounced when more groups were present.

4. The model further predicts an asymptotic relationship between number of functionally analogous species and probability of functioning, rather than a linear or complex one. In case the species are very prone to extinction, saturation occurs at higher species numbers, which indicates there is less species redundancy in vulnerable systems.
Immune defence reaction in bumble‐bee workers after a previous challenge and parasitic coinfection
Authors: Allander K. , Schmid‐Hempel P.
Published: March 2003

A successful immune response requires the functioning of a range of physiological mechanisms and is therefore supposed to be costly in terms of limited resources that could be used otherwise, such as energy or nutrients. Because immunity is costly, immune defence should be reduced if appropriate multiple immunogenic challenges are present.

The working hypothesis that multiple challenges will reduce the immune system’s capacity to respond is adopted. An existing infection by yet another parasite could also further reduce the strength of the immune response, since limited resources have to be divided among the different challenges.

The bumble‐bee, Bombus terrestris L., and its parasites are used as a model system to study (1) whether the melanization response towards an antigen is reduced or increased after a preceding challenge (‘depletion/stimulation’); (2) if depletion occurs, how long it takes to mount a full defence response against the second challenge; and (3) whether the coinfection by another parasite reduces or increases the immune defence system’s capacity to respond. A standard technique was used where two novel antigens (nylon filaments of different sizes and time intervals) were implanted into the haemocoel of individual workers. Controls were individuals implanted with a single fixed sized implant. A gut parasite, the trypanosome Crithidia bombi, was used for coinfections.

Size of the implant had no effect on the melanization response as well as time between primary and secondary implant. Primary and secondary implants differed from the controls. In particular, the primary implant was melanized more intensively than the controls and secondary, respectively. Furthermore, a coinfection by C. bombi had no effect on the encapsulation response.

The results suggest that no general depletion of the encapsulation system occurs under the conditions of our experiment and that a similar immune response (melanization) independent on the magnitude of the challenge, occurs. However, there seems to be a depletion effect on the secondary implant. Furthermore, the dynamics of two different parasites seem to run independently as far as within‐host interactions are concerned.
Changing nutritional status causes a shift in the balance of nocturnal to diurnal activity in European Minnows
Authors: Metcalfe N. B. , Steele G. I.
Published: March 2003

For animals that have access to a predator‐free refuge, the daily timing of feeding activity should reflect temporal variation in both the risk of predation while foraging and the rate of food intake: the preferred period of activity should be that which minimizes the predation risk per unit of food obtained.

 However, several species of freshwater fish show flexibility in the daily pattern of feeding activity. European Minnows Phoxinus phoxinus L. prefer to feed at night at low temperatures, spending the majority of the day hiding in refuges. Since their feeding efficiency is lower at night, this behaviour is thought to minimize exposure to diurnal predators. The fish become increasingly active in the day as the temperature increases, possibly because their food requirements cannot be met by only feeding at night.

 Here, it is tested experimentally whether nutritional state can affect the daily activity schedule, by manipulating the energy reserves of over‐wintering minnows and recording their relative night‐ and daytime use of refuges.

 Well‐nourished fish were often observed hiding in refuges, especially during the day. As their energetic reserves decreased, they were more likely to be seen out of the refuges both by day and by night. However, the change in activity was greater by day, so that the fish became increasingly diurnal as their nutritional reserves declined. This effect was independent of temperature.

 The results support the hypothesis that the fish attempt to minimize their exposure to diurnal predators, but must balance this against the need to increase their level of daytime foraging activity when in energy deficit.
Respiratory carbon loss of calcareous grasslands in winter shows no effects of 4 years’ CO2 enrichment
Authors: Volk M. , Niklaus P. A.
Published: March 2003

CO2 exchange measurements in long‐term CO2‐enrichment experiments suggest large net carbon gains by ecosystems during the growing season that are not accounted for by above‐ground plant biomass. Considerable amounts of C might therefore be allocated below ground.

Winter ecosystem respiration from temperate grasslands under elevated CO2 may account for the loss of a significant part of the extra C gained during the growing season. To test this hypothesis, dark respiration was assessed throughout the winter of the fourth year of CO2 enrichment in a calcareous grassland.

Using these data, a model was parameterized to estimate whole‐winter respiratory CO2 losses. From November to February, 154  9 g C m−2 were respired under elevated CO2 and 144  5 g C m−2 under ambient [CO2], with no significant difference between the CO2 treatments.

We conclude that (i) wintertime respiration does not constitute a larger C loss from the ecosystem at elevated CO2; and (ii) the absence of respiratory responses implies no extra growing‐season C inputs with month‐to‐year turnover times at elevated CO2.
Shade acclimation of rainforest leaves to colonization by lichens
Authors: Anthony P. A. , Holtum J. A. M. , Jackes B. R.
Published: March 2003

Lichens that live epiphytically on leaves (foliicolous lichens) are one of the most abundant groups of epiphytes in tropical rainforests, with lichen cover on individual leaves often exceeding 50%. In this study we quantified the shading of host leaves by foliicolous lichens, and investigated the capacity of palm leaves growing under contrasting light regimes to photo‐acclimate to lichen colonization.

Spectral analyses of 11 lichen species with differing thallus colouring and morphology demonstrated that the lichens exhibited absorptance spectra similar to their host leaves. The lichens showed mean light interceptions of 50%, with some species reducing the photosynthetically usable light by up to 70%.

Despite such shading, our observations indicated that leaves photo‐acclimate to the cover of lichens. The rate of photosynthesis of leaf patches covered by lichens was saturated at lower irradiances, and these patches had higher chlorophyll concentrations than uncolonized leaf areas. Monte Carlo estimates of total daily carbon gain, based on assimilation–irradiance curves and continuous light records, indicated that leaf areas covered by lichens compensated fully for the shading by lichens.

These findings do not support the widely held opinion that shading by foliicolous lichens and other epiphylls generally reduces the rate of photosynthesis by their host leaves.
Phenological and morphological adaptations to the light environment in two woody and two herbaceous plant species
Authors: Kikuzawa K.
Published: March 2003

Leaf emergence, leaf longevity, light regime and photosynthetic rates were investigated in order to clarify the morphological and phenological adaptations to light conditions by avoiding self‐shading in two herbaceous (Polygonatum odoratum and Polygonum sachalinensis) and two woody (Fagus crenata and Alnus sieboldiana) plants in artificially created open habitats in Japan.

Fagus and Polygonatum open their leaves simultaneously as a flush during a short period at the start of the growing season (simultaneous foliar phenology), while Alnus and Polygonum open their leaves one by one successively for a longer period (successive foliar phenology).

Light conditions at the level of single leaves, defined as photon flux density relative to that above the plant, were essentially the same throughout a growing season for simultaneous species. On the other hand, light conditions for the two successive species degraded with time. There were gradients of light from the distal to basal part of a shoot only in successive species.

Reflecting the abrupt decrease in irradiance with time, photosynthetic rates of individual leaves decreased quickly with time in successive species (Alnus and Polygonum), while those of simultaneous species (Fagus and Polygonatum) decreased slowly. Photosynthetic rates of the canopy as a whole, however, were maintained constant over the season in successive species by replacing leaves frequently. Canopy‐level photosynthesis decreased slowly with time in the two simultaneous species.

Successive leaf emergence is adaptive in open habitats such as flood plains and canopy gaps, while simultaneous leafing is adaptive in light‐limited habitats such as the forest understorey.
Reduced leaf expansion as a cost of systemic induced resistance to herbivory
Authors: Moore J. P. , Taylor J. E. , Paul N. D. , Whittaker J. B.
Published: March 2003

We investigated whether increases in cell wall peroxidases link the benefits of induced resistance and the potential ecological costs of reduced leaf expansion.

Rumex obtusifolius is a host plant of the chrysomelid beetle Gastrophysa viridula. The effect of feeding by G. viridula on plant physiology and activity of cell wall‐bound peroxidase activity in the plant was measured under controlled conditions.

Gastrophysa viridula were applied to fully expanded leaf 4, and expansion of leaf 8 was measured over 22 days. Removal of leaf tissue by the herbivore delayed subsequent leaf emergence in treated plants and temporarily reduced the expansion rate of leaf 8, beginning 5 days after treatment. This reduction continued for 4 days when compared with non‐treated plants, and reduced final leaf area by approximately 37%.

Final epidermal cell areas in leaf 8 were reduced by approximately 13%, while epidermal cell numbers remained unchanged.

Cell wall‐bound peroxidase activity was measured in leaf 8 over a 7‐day period following attack. Activity increased within 24 h following the start of treatment and peaked at day 3.

We postulate a herbivore‐induced systemic mechanism linking reductions in cell expansion and subsequent leaf growth rates as a result of an increase in cell wall‐bound peroxidase activity.

Herbivore grazing commonly reduces subsequent host growth, and the assumption has been that this is largely due to the consequences of leaf area lost to grazing. This study suggests that reduced growth, and the associated ecological costs result from induced resistance, and are the consequence of a more subtle direct physiological effect in constraining leaf expansion.
DNA polymerase ε encoded by cdc20+ is required for chromosomal DNA replication in the fission yeast Schizosaccharomyces pombe
Authors: Sugino Akio , Ohara Takeshi , Sebastian Josef , Nakashima Naomi , Araki Hiroyuki
Published: March 2003

Background:

DNA polymerase II (PolII), the homologue of mammalian DNA polymerase ε, is essential for chromosomal DNA replication in the budding yeast Saccharomyces cerevisiae and also participates in S‐phase checkpoint control. An important issue is whether chromosomal DNA replication in other eukaryotes, including the fission yeast Schizosaccharomyces pombe—in which the characteristics of replication origins are poorly defined—also requires DNA polymerase ε. It has been shown that DNA polymerase ε is not required for the in vitro replication of SV40 DNA by human cell extracts.

Results:

We have cloned and sequenced S. pombepol2+, which is identical to the cell‐cycle gene cdc20+, encoding the catalytic polypeptide of DNA polymerase ε (Polε). The predicted amino acid sequence of Polε is highly homologous to that of S. cerevisiae PolII and human Polε. Consistent with this, the Polε polypeptide was recognized by polyclonal antibodies against S. cerevisiae PolII holoenzyme (PolII*). The terminal morphology of cells containing the disrupted pol2 gene was similar to that of DNA replication mutant cells and cdc20 mutant cells. Furthermore, the Polε activity from temperature‐sensitive S. pombecdc20 mutant cells was temperture‐sensitive, and chromosomal DNA replication in the mutant cells was inhibited at the restrictive temperatures.

Conclusion:

These data strongly suggest that Polε is required for normal chromosomal DNA replication in S. pombe, as is PolII in S. cerevisiae. Thus, eukaryotic chromosomal DNA is replicated differently from that of viral SV40 DNA.
Sequence elements that contribute to the degradation of yeast Gα
Authors: Schauber Cherylene , Chen Li , Tongaonkar Prasad , Vega Irving , Madura Kiran
Published: March 2003

Background

Gpa1 is the α subunit of the yeast G‐protein that regulates signal transduction during mating. The stability of Gα/Gpa1 is influenced by the ubiquitin‐dependent N‐end rule pathway, suggesting that the regulation of Gα levels may be important for effective mating response and recovery.

Results

The Gα sequences that confer sensitivity to degradation by the N‐end rule pathway were identified. The insertion of this degradation signal (G1‐Deg) into the ordinarily stable Gpa2 protein conferred proteolytic targeting. We examined Gα degradation under different conditions and found that it was efficiently degraded in haploid and diploid cells, but was stable if it was synthesized prior to expression of the N‐end rule pathway. Interestingly, a specific mutation in Gα that is believed to promote the GTP‐bound form (N388K) caused accelerated degradation.

Conclusion

A region encompassing a putative effector‐binding domain (G1‐Deg) is required for Gα degradation via the N‐end rule pathway. Our studies have shown that Gα is susceptible to proteolysis soon after synthesis. These results are in agreement with the idea that Gα is more unstable in the GTP‐bound form, which is the predominant state of monomeric/free Gα soon after synthesis. It is likely that the signal transduced by Gβγ can be regulated by adjusting the levels of Gα through proteolysis.
Grb10/GrbIR as an in vivo substrate of Tec tyrosine kinase
Authors: Mano Hiroyuki , Ohya Ken‐ichi , Miyazato Akira , Yamashita Yoshihiro , Ogawa Wataru , Inazawa Johji , Ikeda Uichi , Shimada Kazuyuki , Hatake Kiyohiko , Kasuga Masato , Ozawa Keiya , Kajigaya Sachiko
Published: March 2003

Background

Tec is a member of the recently emerging subfamily among nonreceptor protein‐tyrosine kinases (PTKs). Although many members of this family have been shown to be involved in a wide range of cytokine‐mediated signalling systems, the molecular mechanism by which they exert in vivo effects remains obscure. To gain insights into the downstream pathways of Tec, we here looked for Tec‐interacting proteins (TIPs) by using the yeast two‐hybrid screening.

Results

One of TIPs turned out to be Grb10/GrbIR, which carries one pleckstrin homology domain and one Src homology 2 domain. Grb10/GrbIR was known to bind receptor PTKs in a ligand‐dependent fashion, but not to be phosphorylated on tyrosine residues. In a transient expression system in human kidney 293 cells, however, Grb10/GrbIR becomes profoundly tyrosine‐phosphorylated by Tec, but not by Syk, Jak2 or insulin receptor. We also reveal that expression of Grb10/GrbIR suppresses the cytokine‐driven and Tec‐driven activation of the c‐fos promoter.

Conclusion

Our results indicate a novel role of Grb10/GrbIR as an effector molecule to a subset of nonreceptor PTKs.
Crm1 (XpoI) dependent nuclear export of the budding yeast transcription factor yAP‐1 is sensitive to oxidative stress
Authors: Kuge Shusuke , Toda Takashi , Iizuka Narushi , Nomoto Akio
Published: March 2003

Background

The yAP‐1 transcription factor is crucial for the oxidative stress response of the budding yeast Saccharomyces cerevisiae; its activity is induced in response to oxidative stress, and as a consequence the expression of a number of target genes is enhanced. We have shown previously that yAP‐1 is mainly found in the cytoplasm, but that upon the imposition of oxidative stress it localizes to the nucleus. In this study, we addressed the mechanism through which yAP‐1 nuclear localization is regulated.

Results

Here we show that yAP‐1 localization is mediated by active export from the nucleus, resulting from the activity of Crm1 (XpoI), a conserved protein that functions as an export receptor which recognizes the nuclear export signal (NES). When Crm1 expression was repressed, yAP‐1 was localized in the nucleus and induced the expression of a yAP‐1 dependent target gene. Our results also suggest that the cysteine rich domain (CRD), at the C‐terminus of yAP‐1, functions as an export recognition sequence. yAP‐1 and Crm1 interact in vivo and this interaction is reduced in response to oxidative stress.

Conclusions

These results suggest a novel regulatory mechanism of nucleocytoplasmic transport which is dependent upon a redox sensitive nuclear export pathway.
Mice lacking smooth muscle calponin display increased bone formation that is associated with enhancement of bone morphogenetic protein responses
Authors: Yoshikawa Hideki , Taniguchi Shun‐ichiro , Yamamura Hisako , Mori Shigeki , Sugimoto Mizuo , Miyado Kenji , Nakamura Kenji , Nakao Kazuki , Katsuki Motoya , Shibata Nobuhiko , Takahashi Katsuhito
Published: March 2003

Background

Calponin is a calmodulin‐and actin‐binding protein expressed in smooth muscle. It promotes actin polymerization and inhibits actin‐activated myosin ATPase activity. Despite the molecular and functional characterization of calponin in vitro, the physiological role of calponin in vivo has not been clarified.

Results

We investigated the in vivo function of smooth muscle calponin (also called basic calponin or calponin h1) by generating mice carrying a targeted mutation in both alleles of the calponin gene. Mice lacking basic calponin expression displayed enhanced ectopic bone formation in vivo, induced by recombinant human bone morphogenetic protein‐2 (rhBMP‐2), and an augmentation of the degree of osteoblastic differentiation of embryonic mesenchymal cells when they were stimulated by rhBMP‐2. Basic calponin messenger RNA was shown to be expressed in developing and healing bone tissues, and in undifferentiated MC3T3‐E1 osteoblasts. An examination of the skeletons of mutated mice showed an early onset of cartilage formation and ossification, and increased postnatal bone formation characterized by an increase in the number of activated periosteal osteoblasts. Bone fracture healing was accelerated in mutated mice.

Conclusion

This is the first demonstration of animals with enhanced BMP responsiveness in host cells, suggesting that endogenous basic calponin may play a negative role in an osteogenic programme.
Cytoskeletal reorganization by soluble Wnt‐3a protein signalling
Authors: Shibamoto  , Higano Keiichi , Takada Ritsuko , Ito Fumiaki , Takeichi Masatoshi , Takada Shinji
Published: March 2003

Background:

Wnt‐3a is an intercellular signalling molecule that is involved in a variety of morphogenetic events. However, the molecular mechanisms underlying Wnt‐3a signalling are poorly understood. We have sought to establish in vitro systems to assay the activity of this protein and investigate its biological roles.

Results:

We prepared mouse L cells transfected with Wnt‐3a cDNA, and found that their β‐catenin protein level was up‐regulated. When conditioned medium (CM) was collected from cultures of the transfectants and added to nontransfected L cells, the β‐catenin level of the latter was also increased. Approximately 50% of the Wnt‐3a proteins synthesized by the transfectants were secreted into the CM in a soluble form. These secreted Wnt‐3a proteins formed an activity gradient in the environment surrounding the transfectants. Then, we studied whether Wnt‐3a had any effect on cellular behaviour in vitro. When the CM containing Wnt‐3a (W3a‐CM) was added to cultures of C57MG mammary epithelial cells, their morphology was altered to exhibit closer intercellular contacts. Immunostaining for various adhesion and cytoskeletal proteins showed that the actin–microfilamental system was re‐organized by the W3a‐CM treatment. It induced a directional alignment of actin stress fibres and other actin‐associated proteins. Moreover, villin, localized only at the perinuclear regions in untreated C57MG cells, was re‐distributed to the leading edges of the cells, co‐localizing with F‐actin, in the presence of Wnt‐3a.

Conclusion:

Our findings suggest that Wnt‐3a protein, in the soluble form, can act to re‐organize cytoskeletal structures.
Eukaryotic circadian systems: cycles in common
Authors: Dunlap Jay C. , Loros Jennifer J. , Liu Yi , Crosthwaite Susan K.
Published: March 2003

Common regulatory patterns can now be discerned among eukaryotic circadian systems, extending from fungi through to mammals. Complexes of two distinct PAS domain‐containing transcription factors play positive roles in clock‐associated feedback loops by turning on classic clock proteins such as FRQ, PER and TIM. These in turn appear to act as negative elements, interfering with their own activation and thus giving rise to an oscillatory negative feedback loop. Post‐transcriptional control governs the amount and type of FRQ and makes the clock responsive to temperature.
α2D/A‐adrenergic receptor gene induction in the retina by phorbol ester: involvement of an AP‐2 element
Authors: Venkataraman Venkateswar , Duda Teresa , Sharma Rameshwar K.
Published: March 2003

Background

The α2‐adrenergic receptor (α2‐AR) expressed in the bovine retina has been demonstrated to be of the α2D subtype. The bovine α2D‐adrenergic receptor (α2D/A‐AR) gene has been cloned and characterized. This report describes the induction of this gene by phorbol‐ 12, 13‐myristate acetate (PMA), an activator of protein kinase C (PKC).

Results

Treatment of the bovine retina for 60 min with PMA (1 μm) resulted in significant and similar increases in α2D/A‐AR mRNA level and gene transcription. This indicates that PMA causes α2D/A‐AR gene induction and that this induction takes place directly at the transcriptional level. In C6 cells, treatment with PMA at a concentration which was as low as 0.1 μm induced endogenous α2D/A‐AR mRNA after 60 min. Luciferase reporter assays in C6 cells mapped the PMA‐responsive element to a region between −247 bp and −163 bp on the α2D/A‐AR promoter. Electrophoretic mobility shift assays showed an increased binding of nuclear factor(s) from PMA‐treated bovine retina to this promoter region. Competition assays indicate that an AP‐2 element may be involved in the PMA‐dependent induction.

Conclusion

These findings demonstrate for the first time, the direct induction of the α2D/A‐AR gene by PMA and support a role for an AP‐2 element in the induction mechanism.
Protein nucleocytoplasmic transport and its light regulation in plants
Authors: Yamamoto Naoki , Deng Xing‐Wang
Published: March 2003

Light exerts a great influence on gene expression, physiology and development pattern in higher plants. Protein factors involved in these processes, such as the photoreceptor, phytochrome B, a key regulatory protein, COP1, and some bZIP transcription factors have been identified and their light‐regulated movement between the cytoplasm and the nucleus has been demonstrated. These findings imply that nucleocytoplasmic transport plays a crucial role in light regulation in higher plants. This review summarizes recent investigations into plant nuclear transport systems and specific cases where nucleocytoplasmic transport is subject to light regulation.
Regulation of DNA gyrase operon in Mycobacterium smegmatis: a distinct mechanism of relaxation stimulated transcription
Authors: Unniraman Shyam , Nagaraja Valakunja
Published: March 2003

Background

The topological state of DNA is a result of the diverse influences of various topoisomerases present in the cell. Amongst these, DNA gyrase is the only enzyme that is capable of supercoiling DNA. In all the eubacterial cells tested so far, DNA gyrase has proved to be essential for survival. We have earlier cloned gyr genes from Mycobacterium smegmatis. Unlike the situation in Escherichia coli, genes encoding the two subunits of gyrase are present as a contiguous stretch in the M. smegmatis genome.

Results

We have demonstrated that the two subunits are encoded by a single dicistronic message, with the transcriptional start site mapping 57 base pairs upstream of the putative translational start of the gyrB ORF. The gyr promoter is specific to M. smegmatis and does not function in E. coli. We have shown that the synthesis of DNA gyrase in M. smegmatis is induced by novobiocin—a known inhibitor of gyrase. Short fragments encompassing the promoter region, when cloned in a promoter selection vector, do not show any response to changes in supercoil levels. Larger fragments show a supercoil sensitive behaviour, as seen in the genomic context.

Conclusions

The gene structure and the transcriptional organization of the gyr operon suggest an overall regulatory scheme that is unique to mycobacteria. In contrast to E. coli, promoter and regions in its vicinity are not sufficient to confer supercoil sensitivity. Promoter distal regions— 600 bp downstream of the promoter—appear to be necessary for relaxation‐stimulated transcription in M. smegmatis.
Association of Lyn tyrosine kinase to the GM‐CSF and IL‐3 receptor common βc subunit and role of Src tyrosine kinases in DNA synthesis and anti‐apoptosis
Authors: Dahl Martin E. , Arai Ken‐ichi , Watanabe Sumiko
Published: March 2003

Background

After GM‐CSF or IL‐3 stimulation, the activation of JAK2 tyrosine kinase and members of the Src family of tyrosine kinases takes place, followed by phosphorylation of βc tyrosine residues and the recruitment of SH2 containing molecules to the receptor complex. The exact role of Src kinases such as Lyn in this and other downstream signal transduction events remains unclear.

Results

We investigated the association of Lyn kinase with βc using synthetic peptides derived from the eight βc tyrosine residues and the Box 1 motif. We found that Lyn kinase GST fusion proteins bind to peptides corresponding to the membrane proximal region of βc and to peptides containing specific βc derived phosphorylated tyrosine residues. We also determined that βc tyrosine residues Y1,2 as well as Y7 and Y8 can act as substrates of Lyn. We further analysed the role of the Src kinases in DNA synthesis and anti‐apoptosis downstream of GM‐CSF by using the Src kinase inhibitor PP1 in murine BA/F3 cells stably expressing a series of mutant βc receptors.

Conclusions

Lyn binds to βc derived peptides through multiple interactions, and may play an important role in βc phosphorylation. Src family kinases also play an essential role in GM‐CSF mediated DNA synthesis, as well as an important role in anti‐apoptosis in response to GM‐CSF.
Carboxyl‐terminal region conserved among phosphoinositide‐kinase‐related kinases is indispensable for mTOR function in vivo and in vitro
Authors: Takahashi Tetsuya , Hara Kenta , Inoue Hitomi , Kawa Yoshitaka , Tokunaga Chiharu , Hidayat Sujuti , Yoshino Ken‐ichi , Kuroda Yoshikazu , Yonezawa Kazuyoshi
Published: March 2003

Background

The mammalian target of rapamycin (mTOR) belongs to the family of phosphoinositide (PI)‐kinase‐related kinases that includes the ataxia‐telangiectasia gene product (ATM). mTOR plays a critical role in controlling translational effectors such as p70 S6 kinase α (p70α) and eukaryotic initiation factor 4E binding protein 1 (4EBP1).

Results

We show that the C‐terminal region of mTOR, which is highly conserved among the PI‐kinase‐related kinases, plays a critical role in the mTOR protein kinase activity. Deletion of the C‐terminal residues did not adversely affect the expression of mTOR, but caused a nearly complete loss of the mTOR protein kinase activity toward both 4EBP1 and p70αin vitro. These deletions also abolished the ability of a rapamycin‐resistant mTOR mutant to rescue the activity of p70α from inhibition induced by rapamycin in vivo. Furthermore, replacement of Trp2545, a conserved residue in the C‐terminal region throughout the PI‐kinase‐related kinase family, abolished the function of the mTOR kinase, both in vivo and in vitro. However, substitution of 32 C‐terminal residues of mTOR with those of ATM did not restore the mTOR function.

Conclusions

These findings define an indispensable role for the noncatalytic C‐terminal region of mTOR and indicate that, although this highly conserved region may be important throughout the PI‐kinase‐related kinase family, it is not functionally interchangeable within the family.
Two basic residues, Lys‐107 and Lys‐118, of RuvC resolvase are involved in critical contacts with the Holliday junction for its resolution
Authors: Yoshikawa Manabu , Iwasaki Hiroshi , Kinoshita Kengo , Shinagawa Hideo
Published: March 2003

Background

Crystallographic and mutational studies of Escherichia coli RuvC Holliday junction resolvase have revealed that a catalytic site of each subunit is composed of four acidic residues at the bottom of the putative DNA‐binding cleft, whose surface contains eight basic residues.

Results

To elucidate the functional roles of the basic residues on the cleft surface, we constructed a series of mutant ruvC genes and characterized their properties in vivo and in vitro. Among them, two RuvC mutants with a single alteration, K107A and K118A, were defective in UV‐repair and showed a dominant negative effect. The purified K107A and K118A proteins showed reduced binding activity to the junction DNA in the presence of Mg2+ under high salt conditions. Mn2+ increased both the junction binding and cleaving activities of the mutant proteins. In the absence of a divalent cation, the wild‐type, K107A and K118A proteins did not bind to junction DNA under high salt conditions, but the D7N mutant, with an alteration of the catalytic centre, was able to bind to the junction efficiently.

Conclusion

The results presented here, in conjunction with previous crystallographic studies, suggest that the catalytic complex which is formed through interactions of acidic residues, Mg2+ and a cleavable phosphodiester bond, is stabilized by Lys‐107 and Lys‐118 via electrostatic interactions with the DNA backbone, a process which is critically important for the cleavage reaction to take place. One or two basic residues near the catalytic centre have also been found in other RNase H superfamily proteins, indicating that this is the conserved reaction mechanism in this superfamily.