bluuebunny/crossref_metadata_2025 huggingface dataset:
Optimal timing of corolla abscission: experimental study on Erythronium japonicum (Liliaceae)
Authors: Ishii H. S. , Sakai S.
Published: March 2003

1. We analysed how male and female success affects the floral longevity in Erythronium japonicum. We found that flower senescence is advanced by deposition of pollen grains on the stigma but that there exists a minimum longevity (13·64 days in 1996 and 12·20 days in 1997) during which the flower never abscises, even if it is pollinated with a sufficient number of pollen grains.

2. We suggest that this floral longevity does not exist to benefit the female function, because the time needed for corolla abscission reacting to ovule fertilization was not so long and the female function was completed within about 3 days.

3. Flowers of E. japonicum do not react to pollen removal, as shown by the fact that anther removal experiments did not affect floral longevity. However, it would be advantageous to have some duration of longevity which benefits male success such that most of pollen grains can be expected to be exported. The duration needed to disperse most pollen grains of a flower was about 12 days, very close to the minimum floral longevity.

4. We conclude that a minimum longevity may exist for male functions and that floral longevity may be influenced differently by the male and the female functions of flowers.
Escape flight in moulting Tree Sparrows (Passer montanus)
Authors: J. Lind
Published: March 2003

Impaired predator evasion in birds as a cost in different life‐history periods has received increasing attention in the last decade. Evasive abilities in birds have been found to be detrimentally affected by migratory fuel load, reproduction and moult. These results suggest that during these periods of their lives birds suffer from increased predation risk due to impaired evasive abilities.

 Theoretically, moult should have a detrimental effect on flight, and empirical work on starlings has shown impaired escape ability due to moult. However, a recent theoretical investigation found a surprisingly small effect of moult on flight in birds.

 In this study, 31 Tree Sparrows, a sedentary species with a slow moult, were used to investigate the effect of natural and manipulated moult on escape ability. No effect was found due to natural moult, however, when experimentally increasing moult gap size a strong negative effect was found.

  With support from empirical and theoretical work, this is the first study to suggest that slow moult may not increase predation risk due to impaired evasive abilities. Compensatory physiological adaptations probably cause this result and may be very important during moult.

 Predation risk is probably an important factor in the evolution of moult patterns and moult strategies.
Canopy‐level photosynthetic compensation after defoliation in a tropical understorey palm
Authors: N. P. R. Anten , D. D. Ackerly
Published: March 2003

1. Increases in photosynthesis of leaves remaining after defoliation may result from shifts in leaf photosynthetic characteristics or from an improved light penetration in the canopy. However, few studies have tried to estimate the relative contribution of these factors to mitigating the negative effects of defoliation. We present a quantitative framework for such an analysis.

2. In a field and greenhouse experiment, plants of the rain forest understorey palm Chamaedorea elegans were subjected to three levels of defoliation (0, 50 or 66% of leaves removed) and, in the greenhouse, grown at two irradiances (5 or 16% of daylight, ‘low light’ and ‘high light’ hereafter). For each plant, leaf photosynthesis and light penetration in the canopy were measured and these data were used to calculate whole‐canopy carbon gain.

3. Defoliation significantly increased the light available to the remaining leaves. In the field and in the ‘high‐light’ greenhouse plants, defoliation also resulted in an increase in the light‐saturated photosynthesis per unit leaf area (Pmax), although in the ‘low‐light’ plants this was not the case.

4. A sensitivity analysis revealed that in the ‘low‐light’ plants, a proportional increase in leaf Pmax did not result in increased canopy‐level carbon gain, while in the ‘high‐light’ and field plants, it did. This suggests that the lack of plasticity in Pmax in response to defoliation, at low light, may be the appropriate pattern to maximize carbon gain.

5. Defoliated plants had 10–18% more average photosynthesis per unit leaf area (Parea) than the control plants, and this increase was mostly the result of an improved light penetration in the canopy.

6. A relatively small fraction (5–30%) of the total estimated loss in canopy carbon gain caused by the removal of leaves was compensated for by an increase in Parea. This suggests that in rain forest understorey plants, an increase in diurnal photosynthesis of remaining leaves has limited potential for mitigating the negative effects of defoliation.
Fat stores in birds: an overlooked sink for carotenoid pigments?
Authors: Negro J. J. , Figuerola J. , Garrido J. , Green A. J.
Published: March 2003

Carotenoids are responsible for the most striking colours in birds, but also play an important role as enhancers of the immune system. Consequently, a trade‐off between the ornamental and health functions of carotenoids in birds has been proposed.

 Although it is well known that birds can store carotenoids in different organs and tissues, including the fat stores, until now all field studies of the regulation of carotenoid stores have focused on plasma carotenoids.

 Carotenoids in the fat of 44 wild Greylag Geese (Anser anser L.) wintering in south‐western Spain were identified and quantified. In addition, the relationships between carotenoids and the size of the fat stores, as well as the sex and age of the geese, were analysed.

 The major carotenoid in goose fat was lutein. This and related carotenoids are also the most prevalent pigments in bird plasma and secondary sexual characters. We also detected β‐cryptoxanthin, β‐carotene, neochrome and neoxanthin. Total carotenoid concentration was negatively correlated with the size of the fat stores. Males had higher concentrations of carotenoids than females.

A possible explanation for these patterns is that male birds tend to have higher plasma carotenoids than females, a difference that could be transposed to fat if carotenoids diffused passively from the blood into adipose tissues. Carotenoids, however, may tend to remain in the fat stores. If this were true, fat would not be a reservoir of carotenoids, but a sink where a significant amount of these pigments would be sequestered, being no longer available for other functions.
Competition mediates costs of jasmonate‐induced defences, nitrogen acquisition and transgenerational plasticity inNicotiana attenuata
Authors: Van Dam N. M. , Baldwin I. T.
Published: March 2003

Plants adjust their phenotype in response to environmental signals, but little is known about the interaction of plastic responses to simultaneously occurring environmental stresses.

 To examine the costs of induced resistance on reproductive performance in plants subjected to other important environmental variables, resistance was elicited with a jasmonate treatment (MeJA) to one, both or neither of twoNicotiana attenuataplants growing competitively in either high‐ or low‐N soils. Half the plants were subjected to leaf removal (LR). K15NO3was used to quantify differences in N acquisition and allocation. Transgenerational effects were measured with seed germination and seedling performance tests.

 An induced plant competing with an uninduced plant produced significantly fewer seeds, acquired less15N and allocated less15N to seed production. Uninduced plants competing with induced plants realized a comparable fitness benefit.

 The costs of induction were greater under high N. Plants grown under low N minimized costs by allocating significantly more N to seeds. LR decreased seed production independently of any other effect. Low N and LR both reduced germination rates.

 The effects of MeJA on seed germination depended on competition and N supply. The differences in germination rates resulted in dramatic fitness differences among offspring.

 N. attenuataplants appear to use N availability and their induced status to alter their current phenotype and their offspring’s phenotype to adjust to environmental changes that occur predictably over time in their natural environment.
Metabolic rate and aggressiveness between Brown Trout populations
Authors: Lahti K. , Huuskonen H. , Laurila A. , Piironen J.
Published: March 2003

Studies focusing on the physiological variation between populations and its connection to fitness‐related traits are rare, even though integrating these fields would increase knowledge on the evolution of traits. Standard metabolic rate (SMR) has been suggested to influence an individual’s social status and the level of aggressiveness, as dominant individuals tend to have higher SMR than subordinate individuals.

 The SMR of juvenile Brown Trout (Salmo trutta) from four populations was measured, and the population‐level relationship between SMR and aggressiveness, which is a fitness‐related behavioural trait, was investigated.

 SMR differed between the populations, but no differences were found in the amount of aggression. Nevertheless, a significant positive correlation between aggressiveness and SMR between the populations was found.

 Unlike many previous studies on geographical variation of metabolic rate, SMR correlated negatively with the latitude of origin of the populations.

 The results suggest that SMR and aggressiveness are correlated not only at the individual level as shown by previous studies, but also at the population level. The costs and benefits of high metabolic rate depend largely on the environment, and local differences in environmental conditions, as for example in food availability, may select for local differences in SMR.
Does temperature limit the invasion of Impatiens glandulifera and Heracleum mantegazzianum in the UK?
Authors: Willis S. G. , Hulme P. E.
Published: March 2003

1. Impatiens glandulifera Royle and Heracleum mantegazzianum Sommier et Levier are widespread, non‐indigenous plant species in the UK. A variety of correlational analyses suggest that their spatial extent is limited by climate, although no experimental studies have tested this hypothesis. This paper reports the first detailed experimental examination of the impact of climate on the performance of the two species.

2. Seeds of each species were sown, in each of 2 years, in replicated plots along an elevational gradient (10–600 m a.s.l.) in north‐east England. Both species germinated readily at all elevations, even in areas well above their current limits within the study area. The plants were, however, smaller at higher altitudes. Impatiens glandulifera also produced fewer seeds with increasing elevation.

3. Plant performance was assessed in relation to actual and interpolated climate data along the elevational transect. For H. mantegazzianum, the timing of germination was correlated most strongly with the pre‐emergence heat sum; for I. glandulifera this relationship was significant in one year only. Maximum height of both species was correlated with increasing post‐emergence heat sum, as was pod production by I. glandulifera. The biomass of second‐year H. mantegazzianum plants varied non‐linearly with post‐emergence heat sum. For both species, overwinter survival of seeds was not related to winter temperature or frost days. Overwinter survival of first‐year H. mantegazzianum plants declined with increasing frost incidence.

4. The results suggest that, of the two species, only I. glandulifera is currently most limited by temperature, although this is not the only factor determining the distribution of the species.
In situ water absorption by tree fine roots measured in real time using miniature sap‐flow gauges
Authors: Coners H. , Leuschner C.
Published: March 2003

Root water uptake is a key process in the circulation of water in forest ecosystems. Until recently, water absorption by tree fine roots could not be measured in situ in undisturbed soil.

We present a new technique that allows continuous recording of the water absorption of fine root endings in mature stands without altering soil structure, hydrology or mycorrhizal infection.

The approach combines miniature sap‐flow gauges mounted on small‐diameter tree roots (3–4 mm) with a complete extraction and visual surface analysis of the adjacent absorbing fine root endings. This technique yields continuous data on water absorption per fine root surface area, and allows analysis of the spatial heterogeneity of root water uptake in the rhizosphere of forests.

We present the results of laboratory and field calibration experiments with Fagus sylvatica L. roots (3–4 mm), which show a good agreement between gauge flow data and synchronous gravimetric flow measurements for flows between 2 and >50 g h−1. Gauge readings were unreliable during low flows (<2 g h−1) at night. In these periods, which cover ≈10% of daily flow, we used an empirically derived linear relationship between root temperature difference and flow.

Measurements on F. sylvatica root endings during 10 summer days showed daily water absorption maxima ranging between 0·20 (rainy days) and 0·58 mmol m−2 root surface area s−1 (bright or overcast days). The corresponding daily maxima of leaf transpiration rate were ≈10 times higher (2–4 mmol m−2 leaf area s−1).

The combination of miniature sap‐flow gauges and determination of fine root surface area provides a promising tool for analysing water absorption by tree root systems in situ.
Predicted and observed maximum prey size – snake size allometry
Authors: King R. B.
Published: March 2003

For gape‐limited predators such as snakes, it should be possible to predict the relationship between maximum prey size and body size from the relationship between maximum prey size and gape size and between gape size and body size. Such predictions were generated for Water Snakes, Nerodia sipedon L., using a data subset and then tested with a larger data set.

Gape size was computed based on jaw length and width and cyclical regression was used to identify prey of maximum size for snakes of a given gape or mass.

Predicted and observed maximum prey cross‐section–snake mass allometry were in good agreement. Predicted maximum prey mass–snake mass allometry somewhat exceeded observed allometry which did not differ from 1.

Observed minimum prey size–snake size allometry was significantly greater than 0, indicating that larger snakes drop small prey from their diets.

Gape size–body size allometry in two other natricine snakes (Thamnophis sirtalis, Storeria dekayi) suggest that patterns of ontogenetic change in prey size should differ among species in predictable ways.

Sex differences in gape size–snake size allometry suggest that sex differences in maximum prey size should increase with increasing snake size, even when linear measures of head dimensions do not.
Senescence and food limitation in a slowly ageing spider
Authors: Moya‐Laraño J.
Published: March 2003

Evidence for reproductive senescence in invertebrate natural populations is scant probably because most groups are short‐lived or because they lack natural markers of age.

Lycosa tarantula (L.) (the Mediterranean Tarantula) (Araneae, Lycosidae) is a slowly ageing burrowing wolf spider, in which females can reproduce for two consecutive seasons. Females in their first reproductive season (1Y) can easily be distinguished from females in their second reproductive season (2Y) because the lack of pilosity in the latter.

The diet of 1Y and 2Y females was supplemented and their reproductive performance was compared with that of control, non‐food supplemented females. The predictions were that senescent 2Y females would show a worse reproductive performance than 1Y females, and that they would not be able to improve their performance relative to 1Y after food supplementation.

The predictions were met. Older females gained less mass, laid smaller egg sacs, produced fewer spiderlings and, if food supplemented, invested a smaller fraction of their mass in egg sacs. Although 2Y females foraged less actively than younger females, 2Y did not improve their performance relative to 1Y following food supplementation. This pattern of changes provides evidence for reproductive senescence in a natural spider population.
Sendai virus C proteins are categorically nonessential gene products but silencing their expression severely impairs viral replication and pathogenesis
Authors: Kurotani Atsushi , Kiyotani Katsuhiro , Kato Atsushi , Shioda Tatsuo , Sakai Yuko , Mizumoto Kiyohisa , Yoshida Tetsuya , Nagai Yoshiyuki
Published: March 2003

Background:

The P/C mRNA of Sendai virus (SeV), a prototypic member of the family Paramyxoviridae in the Mononegavirales superfamily comprising a large number of nonsegmented negative strand RNA viruses, encodes a nested set of accessory proteins, C′, C, Y1 and Y2, referred to collectively as C proteins, initiating, respectively, at ACG/81 and AUGs/114, 183, 201 in the +1 frame relative to the ORF of phospho (P) protein, the smaller subunit of RNA polymerase. Among them, C is the major species expressed in infected cells at a molar ratio which is several‐fold higher than the other three. However, their function has remained an enigma. It has not even been established whether or not the C proteins are essential for viral replication. Many other viruses in Mononegavirales encode C‐like proteins, but their roles also remain to be defined.

Results:

By taking advantage of a recently developed reverse genetics system to recover infectious SeV from cDNA, we created mutants in which C protein frames were variously silenced. C/C′(−) viruses which did not express C and C′, but did express Y1 and Y2, were severely attenuated in replication in tissue culture cells of various species and tissues, as well as in embryonated chicken eggs. More notably, they were almost totally incapable of growing productively in—and hence nonpathogenic for mice—the natural host. Both gene expression and genome replication appeared to be impaired in C/C′(−) viruses. Additionally silencing the Y1 and Y2 expression was also possible, and a critically impaired but viable clone, the 4C(−) virus, was isolated which expressed none of the four C proteins.

Conclusion:

SeV C proteins are categorically nonessential gene products, but greatly contribute to full replication capability in vitro and are indispensable for in vivo multiplication and pathogenesis. This study represents the first comprehensive functional assessment of the accessary C protein for Mononegavirales.
The mediator for stringent control, ppGpp, binds to the β‐subunit of Escherichia coli RNA polymerase
Authors: Chatterji Dipankar , Fujita Nobuyuki , Ishihama Akira
Published: March 2003

Background

Inhibition of transcription of rRNA in Escherichia coli upon amino acid starvation is thought to be due to the binding of ppGpp to RNA polymerase. However, the nature of this interaction still remains obscure.

Results

Here, the azido‐derivative of ppGpp was synthesized from azido‐GDP and [γ‐32P]ATP by way of the phosphate transfer reaction of the RelA enzyme. The product was subsequently characterized by one and two‐dimensional chromatography. The resulting compound [32P]azido‐ppGpp, where the azido group is attached to the base moiety, was purified to homogeneity and was photo‐crosslinked to Escherichia coli RNA polymerase. SDS‐PAGE analysis of the azido‐ppGpp‐bound enzyme, tryptic digestion and Western blot analysis suggested that azido‐ppGpp binds to the β‐subunit of RNA polymerase.

Conclusion

It was observed that both the N‐terminal and C‐terminal domains of the β‐subunit were labelled with azido‐ppGpp in the native enzyme. However, under denaturing conditions only the C‐terminal part from amino acid residue 802 to residue 1211/1216/1223 was predominantly crosslinked to azido‐ppGpp. The excess of unlabelled ppGpp competes with azido‐ppGpp for binding to the enzyme. azido‐ppGpp inhibits single‐round transcription at the stringent promoter like rrnBP1. In addition, ribosomal protein genes were also found to be inhibited by N3ppGpp. On the other hand, transcription at the lac UV5 promoter remained unaffected upon the addition of azido‐ppGpp.
Synergistic regulation of inducible nitric oxide synthase gene by CCAAT/enhancer‐binding protein β and nuclear factor‐κB in hepatocytes
Authors: Sakitani Kazushige , Nishizawa Mikio , Inoue Kyoichi , Masu Yasuo , Okumura Tadayoshi , Ito Seiji
Published: March 2003

Background

Nitric oxide (NO) has diverse activities under physiological and pathophysiological conditions in many types of cells. In cultured hepatocytes, NO is produced by inducible NO synthase (iNOS) in response to interleukin (IL)‐1β. Cis‐controlling elements and transcription factors which were involved in iNOS gene expression in hepatocytes have been unclear.

Results

We measured the transcriptional activity of the human iNOS gene promoter fused to the firefly luciferase gene in primary cultured rat hepatocytes. The luciferase assay of 5′ deleted promoters revealed that the region from −365 to the transcription initiation site is required for the promoter activity of the iNOS gene. Mutations of a CCAAT/enhancer‐binding protein (C/EBP)‐binding site, namely the A‐activator‐binding site (AABS), and a nuclear factor (NF)‐κB‐binding site within this region, markedly decreased the promoter activity. Transfection of C/EBPβ liver‐enriched activator protein (LAP) or NF‐κB (RelA + p50) activated the iNOS promoter, and transfection of LAP and NF‐κB further activated it synergistically. In addition, either mutation of AABS and the NF‐κB‐binding site markedly reduced the basal promoter activity and the transactivation by LAP, NF‐κB, and a combination of LAP and NF‐κB. Electrophoretic mobility shift assays showed that C/EBPβ was bound to AABS.

Conclusion

These results demonstrate that C/EBPβ may involve iNOS gene expression synergistically with NF‐κB in primary cultured rat hepatocytes.
A core region of the mafK gene IN promoter directs neurone‐specific transcription in vivo
Authors: Motohashi Hozumi , Ohta Jun , Douglas Engel James , Yamamoto Masayuki
Published: March 2003

Background:

MafK serves as a required subunit of erythroid transcription factor NF‐E2 and also functions with various heterodimeric CNC family proteins. MafK expression begins in early mesoderm and is observed in mesenchymal and haematopoietic cells, as well as in neurones during mouse development. In mesodermal descendants, MafK mRNA begins with a distal first exon (called IM), whereas the mRNA in neurones begins with a proximal first exon (IN).

Results:

To elucidate the mechanisms that underlie the tissue‐specific transcription of the mafK gene, and to gain insights into the functions of MafK during neural development, we analysed the activity of the mafK IN promoter. A detailed investigation of mafK expression in the embryonic spinal cord revealed that IN‐initiated mRNA is expressed in the ventral side of the spinal cord. Transient transfection analysis of reporter plasmids bearing the IN promoter and upstream regions revealed that the ‘core’ region of this promoter (nt −67 to −9) is active and that its integrity is crucial for this activity. The core region was also capable of directing the tissue‐specific transcription of a reporter gene in neural cells of the spinal cord in transgenic mice in vivo.

Conclusion:

These results demonstrate that the specific expression of mafK in neural cells is determined, at least in part, by the core region of the IN promoter.
Cloning and characterization of two neural‐salient serine/arginine‐rich (NSSR) proteins involved in the regulation of alternative splicing in neurones
Authors: Komatsu Masaaki , Kominami Eiki , Arahata Kiichi , Tsukahara Toshifumi
Published: March 2003

Background

In neurones, alternative splicing regulates the functions of many gene products. However, the molecular basis of neural‐specific splicing, and how splicing regulation is modulated in different neurones remains to be determined.

Results

We cloned two new SR proteins, Neural‐salient SR proteins (NSSR) 1 and 2, which are present at higher levels in brain and testis. During the differentiation, NSSR 1 is detected only in the neuronal stage. Both the purified recombinant NSSR 1 and 2 proteins enhance the in vitro splicing activity of nuclear extract. Moreover, recombinant NSSR 1 protein enhances the assembly of ribonucleoprotein complexes with S100 fraction. Over‐expression of NSSR 2 prevents the inclusion of either the Flip or Flop exons in the splicing of the GluR‐B gene, resulting in an increase in the abnormal exon‐skipping product. In contrast, transient transfection with NSSR 1 promotes the inclusion of the Flip exon so that the abnormal product is spliced to the mature spliced form. This suppression of exon skipping by NSSR 1 is observed even with co‐transfection of NSSR 2.

Conclusions

NSSR 1 and 2 were cloned from mouse cDNA libraries. Results indicate that NSSR 1 may play a crucial role in the regulation of alternative splicing in neurones.
Molecular mechanism of a cross‐talk between oestrogen and growth factor signalling pathways
Authors: Kato Shigeaki , Masuhiro Yoshikazu , Watanabe Michiko , Kobayashi Yoko , Takeyama Ken‐ichi , Endoh Hideki , Yanagisawa Junn
Published: March 2003

Oestrogen (E2) plays significant roles in variety of biological events such as the development and maintenance of female reproductive organs, bone and lipid metabolisms. More recently, from study of knock‐out mice deficient in oestrogen receptor (ER) α and ERβ it turned out that normal spermatogenesis requires the E2 actions. Furthermore, this female steroid hormone is also well known to be deeply involved in many pathophysiological events such as osteoporosis and cancer development in female reproductive organs. It is particularly well known that most breast cancer is dependent on E2 in its development. Such E2 actions are thought to be mediated through two subtypes of ERs. Growth factors have been shown to synergize in this E2 signalling pathway, although the actual molecular mechanism largely remains unknown.

Recently, we found that the MAP kinase activated by growth factors phosphorylates the Ser118 residue of the human ERα A/B domain and this phosphorylation potentiates the N‐terminal transactivation function (AF‐1) of human ERα, indicating the possible molecular mechanism of a novel cross‐talk between E2 and growth factor signalling pathways. More recently, we have identified a coactivator associating with the hERα AF‐1 in a MAPK‐mediated phosphorylation‐dependent manner. In this review, the molecular mechanism of this cross‐talk is discussed in terms of the transactivation function of ERs, and their coactivators.
The colorectal tumour suppressor APC is present in the NMDA‐receptor‐PSD‐95 complex in the brain
Authors: Yanai Hiroyuki , Satoh Kiyotoshi , Matsumine Akihiko , Akiyama Tetsu
Published: March 2003

Background

The synaptic protein PSD‐95/SAP90 interacts with ion channels such as the N‐methyl‐ D‐aspartate‐receptor (NMDA‐R) via its PDZ domain, and is involved in their clustering. Moreover, it interacts with signalling molecules and plays an important role in coupling NMDA‐R to pathways that control synaptic plasticity and learning.

Results

We report that PSD‐95 interacts with the adenomatous polyposis coli (APC) tumour suppressor protein via its PDZ domain. Furthermore, we found that PSD‐95, NMDA‐R and APC are contained in the same complex in vivo. PSD‐95‐NMDA‐R–APC association was found to require two cysteine residues conserved in the amino‐terminus of PSD‐95 that are known to be critical for its multimerization.

Conclusion

Our findings suggest that the PSD‐95‐NMDA‐R‐APC complex forms due to the multimerization of PSD‐95 monomers, each of which can associate with either NMDA‐R or APC. It is possible that APC is involved in the regulation of ion channel clustering and/or organization of signalling molecules.
Localized phosphorylation of vimentin by Rho‐kinase in neuroblastoma N2a cells
Authors: Nakamura Yu , Hashimoto Ryota , Amano Mutsuki , Nagata Koh‐ichi , Matsumoto Naohiko , Goto Hidemasa , Fukusho Eriko , Mori Hiroshi , Kashiwagi Yujiro , Kudo Takashi , Inagaki Masaki , Takeda Masatoshi
Published: March 2003

Background

Vimentin, which is one of the intermediate filaments, is the major cytoskeletal component in developing neurones or neuroblastoma cells. Rho‐associated kinase (Rho‐kinase), is rich in neurones and is found downstream of Rho. It is involved in the agonist‐induced neurite retraction of neuronal cells, and phosphorylates vimentin at Ser‐38 and Ser‐71 resulting in in vitro disassembly of the filaments.

Results

We have investigated the distribution of vimentin phosphorylated by Rho‐kinase in N2a neuroblastoma cells using site‐specific phosphorylation‐dependent antibodies. TM71 immunoreactivity, which specifically indicates Ser‐71 phosphorylation on vimentin, was found in some neurites of dibutyryl cAMP‐differentiated N2a cells. Transfection of the constitutively active form of Rho‐kinase, CAT, significantly elevated TM71 immunoreactivity, and induced neurite retraction or cell rounding. Conversely, transfection of the dominant negative form of Rho‐kinase, RB/PH(TT), or treatment of 10 μM Y‐27632, a Rho‐kinase specific inhibitor, abolished TM71 immuno‐reactivity, and induced irregular neurite outgrowth. In contrast, 20 n
M okadaic acid (OA) induced neurite retraction and specifically elevated TM71 immunoreactivity. In the OA‐induced neurite retraction, tubulin disappeared in retracting neurites, where vimentin and actin remained co‐localized. Furthermore, the OA‐induced elevation of TM71 immunoreactivity and neurite retraction were completely blocked by pretreatment with 10 μM Y‐27632, or by the ectopic expression of RB/PH(TT).

Conclusions

This study suggests that the localized phosphorylation of vimentin by Rho‐kinase in neurites was closely related with the cellular morphology of N2a cells, and that the Rho‐kinase activity towards vimentin was balanced with OA‐sensitive phosphatases.
MSI‐1, a neural RNA‐binding protein, is involved in male mating behaviour in Caenorhabditis elegans
Authors: Yoda Akinori , Sawa Hitoshi , Okano Hideyuki
Published: March 2003

Neural RNA‐binding proteins are thought to play important roles in neural development and the functional regulation of postmitotic neurones by mediating post‐transcriptional gene regulation. RNA‐binding proteins belonging to the Musashi family are highly expressed in the nervous system; however, their roles are poorly understood.

We identified a Caenorhabditis elegans Musashi homologue, MSI‐1, whose RNA‐recognition motifs show extensive similarity to those of Drosophila and vertebrate Musashi proteins. We isolated a msi‐1 mutant and found males with this mutation to have a mating defect. C. elegans male mating behaviour includes a distinct series of steps: response to contact, backing, turning, vulva location, spicule insertion, and sperm transfer. msi‐1 is required for the turning and vulva location steps. Like other Musashi family members, MSI‐1 is expressed specifically in neural cells, including male‐specific neurones required for turning and vulva location. However, msi‐1 was not expressed in proliferating neural progenitors in C. elegans, unlike the Musashi family genes in other systems.

Our results suggest that msi‐1 is expressed specifically in postmitotic neurones in C. elegans. msi‐1 is required for full development of male mating behaviour, possibly through regulation of msi‐1 expressing neurones.
Allele‐specific detection of nascent transcripts by fluorescence in situ hybridization reveals temporal and culture‐induced changes in Igf2 imprinting during pre‐implantation mouse development
Authors: Ohno Mizuki , Aoki Nao , Sasaki Hiroyuki
Published: March 2003

Background

Genomic imprinting causes parental‐origin‐specific monoallelic transcription of a subset of mammalian genes in the embryo and adult. There is conflicting evidence, however, for the monoallelic transcription of some imprinted genes, such as Igf2, in pre‐implantation embryos.

Results

We have developed an allele‐specific fluorescence in situ hybridization method which involves a pair of oligonucleotide probes designed to detect an intronic polymorphism. The method, called ASO‐RNA‐FISH, enabled us to distinguish allelic nascent Igf2 transcripts in the cell nuclei of early mouse embryos, avoiding signals from the stored oocyte‐specific transcripts. Igf2 transcription was first detectable in two‐cell embryos, and biallelic transcription was predominant up to the morula stage. Then, the maternal allele became silenced during the blastocyst stage. When embryos were cultured in vitro, however, a strong bias to maternal transcription was observed up to the morula stage.

Conclusion

ASO‐RNA‐FISH revealed that a transition of Igf2 from biallelic to monoallelic transcription occurs in the blastocyst stage. This developmental regulation was modified temporarily by in vitro culture, suggesting a possible link between altered imprinting and abnormalities of the foetuses experienced in vitro culture. ASO‐RNA‐FISH is therefore a powerful technique for the study of allele‐specific gene expression.
Sendai virus C protein physically associates with Stat1
Authors: Takeuchi Kenji , Komatsu Takayuki , Yokoo Junko , Kato Atsushi , Shioda Tatsuo , Nagai Yoshiyuki , Gotoh Bin
Published: March 2003

Background The P/C gene of the Sendai virus (SeV), a member of the family Paramyxoviridae, encodes C protein, which plays a crucial role in counteracting the antiviral effect of interferon (IFN). The C protein blocks IFN signalling to prevent the activation of IFN stimulated genes. However, its underlying molecular mechanism remains to be defined.

Results
Signal transducer and activator of transcription 1 (Stat1) is a critical component of IFN‐α/β and IFN‐γ signalling. We found that both unphosphorylated Stat1 and tyrosine‐phosphorylated (pY) Stat1 were present in a form of aberrant high molecular weight complexes (HMWCs) of over 2 MDa in infected cell extracts under low‐salt conditions. Of recombinant vaccinia viruses carrying each SeV gene, only those expressing the C gene induced Stat1‐HMWC. SeV infected cell extracts further displayed an in vitro ability to convert the pY‐Stat1 homodimer to pY‐Stat1‐HMWC. This cell extract activity was not seen after removal of the C protein from the extracts. C protein was therefore involved in the formation of HMWCs. The HMWCs decomposed into smaller complexes in a high‐salt buffer, and under this stringent (high‐salt) condition, as well as a physiological (isotonic) condition, both unphosphorylated Stat1 and pY‐Stat1 were co‐precipitated with anti‐C antibody.

Conclusion
The C protein physically associates with Stat1. This suggests that SeV C protein directly targets Stat1 for inhibitory control on the transcriptional activation of IFN stimulated genes.
Complex phenotype of mice homozygous for a null mutation in the Sp4 transcription factor gene
Authors: Göllner Heike , Bouwman Peter , Mangold Monika , Karis Alar , Braun Harald , Rohner Iris , Del Rey Adriana , Besedovsky Hugo‐Oskar , Meinhardt Andreas , Van Den Broek Marion , Cutforth Tyler , Grosveld Frank , Philipsen Sjaak , Suske Guntram
Published: March 2003

Background Sp4 is a zinc finger transcription factor which is closely related to Sp1 and Sp3. All three proteins recognize the same DNA elements and can act as transcriptional activators through glutamine‐rich activation domains. Unlike Sp1 and Sp3, which are ubiquitous proteins, Sp4 is highly abundant in the central nervous system, but also detectable in many other tissues.

Results We have disrupted the mouse Sp4 gene by a targeted deletion of the exons encoding the N‐terminal activation domains. Sp4 knockout mice show a complete absence of Sp4 expression. They develop until birth without obvious abnormalities. After birth, two‐thirds die within 4 weeks. Surviving mice are growth retarded. Male Sp4null mice do not breed. The cause for the breeding defect remains obscure since they show complete spermatogenesis. In addition, pheromone receptor genes in the vomeronasal organ appear unaffected. Female Sp4null mice have a smaller thymus, spleen and uterus. In addition, they exhibit a pronounced delay in sexual maturation.

Conclusions The phenotype of the Sp4null mice differs significantly from those described for Sp1–/– and Sp3–/– mice. Thus, the structural similarities, the common recognition motif and the overlapping expression pattern of these three transcription factors do not reflect similar physiological functions.
HscA is involved in the dynamics of FtsZ‐ring formation in Escherichia coli K12
Authors: Uehara Tsuyoshi , Matsuzawa Hiroshi , Nishimura Akiko
Published: March 2003

Background FtsZ, a homologue of eukaryotic tubulin, localizes throughout the cytoplasm in non‐dividing Escherichia coli. However, it assembles in cytokinetic rings at the early stages of septation. Factors controlling the dynamics of FtsZ ring formation are unknown, and the molecular mechanism governing these dynamics is yet to be determined.

Results At 42 °C, JE10715 mutant bacteria formed multinucleated filaments with a highly reduced number of FtsZ‐rings at potential division sites. The JE10715 phenotype resulted from a mis‐sense mutation in the hscA gene which encodes a heat shock Hsp70 family protein, with a single alanine‐to‐valine substitution at position 192 within the ATPase domain. Both JE10715 and the hscA knockout strain of JE10715 were completely complemented by a plasmid‐born, wild‐type hscA gene, but not by a mutant‐type hscA715 gene. An hscA conditional knockout of the wild‐type strain under non‐permissive conditions exhibited longer rod cells with an abnormal localization of FtsZ. The over‐expression of dnaK partially complemented the JE10715 mutation. In vitro, the ATPase activity of the mutant protein HscA715 was reduced to 63% of wild‐type HscA activity. HscA co‐sedimented with FtsZ‐polymers in the presence of GTP.

Conclusion HscA is involved in FtsZ‐ring formation, through a chaperon‐like interaction with FtsZ. Defects in hscA, however, can partially be compensated for by redundant genes, including the wild‐type dnaK.
Time course analysis of precocious separation of sister centromeres in budding yeast: continuously separated or frequently reassociated?
Authors: Goshima Gohta , Yanagida Mitsuhiro
Published: March 2003

Background Sister kinetochores are bioriented toward the spindle poles in eukaryotic metaphase before chromosome segregation. In the budding yeast Saccharomyces cerevisiae, sister centromeres/kinetochores are separated in the early spindle, while the sister arms remain associated. Biorientation is thought to be established in this organism with precocious separation of sister centromeres in early stages of the cell cycle. It is not, however, settled whether this pre‐anaphase separation is continuous or only transient and whether the transient separation has any physiological significance.

Results Time‐lapse observation of the behaviour of budding yeast centromeres in living cells was performed using GFP alone or in combination with CFP marking. Sixty‐three per cent of the cell population showed permanent separation of centromeres for a long period of time from the small‐budded stage to the onset of anaphase in the single‐colour GFP‐CEN construct. The remaining cell population (6 of 16) showed brief apparent reassociation of centromere signals before anaphase, but the frequency of the association was very low. In a time‐lapse observation of the double‐colour marked cells by GFP‐CEN and CFP‐SPB (the spindle pole body), the continuous separation of sister centromeres in the short medial spindle was firmly established.

Conclusions In the budding yeast, once sister centromeres separate, they rarely reassociate in pre‐anaphase. Sister centromere cohesion at this stage appears to be irrelevant for normal chromosome segregation. Whether abundant cohesin in the centromere regions has any role in anaphase remains to be determined.
Expression of human and mouse genes encoding polκ: testis‐specific developmental regulation and AhR‐dependent inducible transcription
Authors: Ogi Tomoo , Mimura Junsei , Hikida Masaki , Fujimoto Hirokazu , Fujii‐Kuriyama Yoshiaki , Ohmori Haruo
Published: March 2003

Backgrounds Human polκ is a newly identified low‐fidelity DNA polymerase. While the enzyme bypasses an abasic site and acetylaminofluorene‐adduct in an error‐prone manner, it bypasses benzo[a]pyrene‐N2‐dG lesions in a mostly error‐free manner by incorporating predominantly dC opposite the bulky lesions. Benzo[a]pyrene (B[a]P) is activated through intracellular process mediated by the arylhydrocarbon receptor (AhR, also called the dioxin receptor), which is a ligand‐activated transcription factor with high affinities for aromatic compounds such as B[a]P and dioxin.

Results We examined promoter structures of the human POLK and mouse Polk genes to study how their expressions are regulated. The mouse Polk gene is developmentally regulated in testis and utilizes two transcription start sites during spermatogenesis, while it utilizes only one site in tissues other than testis. Both of the mouse Polk and the human POLK genes have two AhR‐binding sites in the promoter regions and the expression of the mouse Polk gene is indeed enhanced upon AhR‐activation.

Conclusions The AhR activation increases expression of the mouse Polk gene and probably the human POLK gene, the product of which bypasses benzo[a]pyrene‐N2‐dG lesions in a mostly accurate manner. Thus, polκ seems to function to reduce mutagenesis at benzo[a]pyrene‐adducts, although it may also have a role related to spermatogenesis.
Characterization of GTPase‐activating proteins for the function of the Rho‐family small GTPases in the fission yeastSchizosaccharomyces pombe
Authors: Nakano Kentaro , Mutoh Tadashi , Mabuchi Issei
Published: March 2003

BackgroundThe small GTPase Rho1 has been shown to regulate the organization of the actin cytoskeleton and formation of the cell wall in the fission yeastSchizosaccharomyces pombe. Activity of Rho1 must be precisely regulatedin vivo, since both increases and decreases in its activity affect cell growth and shape. Thus, it is important to clarify the mechanism by which the activity of Rho1 is regulatedin vivo.

ResultsSeven genes encoding putative GAPs, GTPase‐activating proteins, for the function of the Rho‐family proteins were isolated fromS. pombe. After disruption of these genes,rga1+was found to play important roles in cell growth and morphogenesis. Inrga1null cells, delocalized F‐actin patches and extraordinary thickening of the cell wall and the septum were observed. On the other hand, over‐expression of Rga1 produced shrunken or dumpy cells. The phenotype of therga1null cells or the Rga1‐over‐expressing cells was similar to that of cells containing abnormally high or low Rho1 activity, respectively. Moreover, direct association of Rga1 with Rho1 was shown. Rga1 was localized to the cell ends and septum where Rho1 is known to function.

ConclusionsInS. pombe, Rga1 is involved in the F‐actin patch localization, cell morphogenesis, regulation of septation, and cell wall synthesis, probably functioning as a GAP for the function of Rho1.
Over‐expression of PAR‐3 suppresses contact‐mediated inhibition of cell migration in MDCK cells
Authors: Mishima Aki , Suzuki Atsushi , Enaka Makiko , Hirose Tomonori , Mizuno Keiko , Ohnishi Tetsuo , Mohri Hiroshi , Ishigatsubo Yoshiaki , Ohno Shigeo
Published: March 2003

Background: PAR‐3 is one of the PAR proteins, previously named ASIP, which are indispensable for the establishment of cell polarity in the embryo as well as differentiated epithelial cells. In mammalian epithelial cells, it forms a ternary complex with aPKC and PAR‐6, and is localized to the tight junction that has been suggested as being important for creating cell polarity.

Results: To gain insights into the mode of PAR‐3 function in mammalian epithelial cells, we examined the effect of PAR‐3 over‐expression in MDCK cells. Although exogenous PAR‐3‐expression does not affect the epithelial polarity of confluent cells, it drastically transforms the morphology of cells at low density into a fibroblastic form with developed membrane protrusions. Time‐lapse observations have revealed that PAR‐3 over‐expressing cells show intense motility, even after they have assembled into loose colonies, suggesting that the contact‐mediated inhibition of cell migration (CIM) is suppressed. The expressions of E‐cadherin and vimentin do not change with PAR‐3 over‐expression, suggesting that exogenous PAR‐3 only disturbs the endogenous equilibrium of cellular states between a fundamental fibroblastic structure and an epithelial one. The co‐expression of a dominant negative mutant of Rac1 and the addition of nocodazole strongly antagonize the effect of PAR‐3 over‐expression, suggesting the involvement of Rac1 activation and microtubule polymerizations.

Conclusions:

The data presented here suggest an intriguing link between the contact‐mediated inhibition of cell migration and the regulation of cell polarity. The putative PAR‐3 activities demonstrated here may function endogenously in the epithelial cell polarization process by being sequestered from the cytosol to the cell‐cell junctional regions with aPKC and PAR‐6 upon cell‐cell adhesion.
Characterization of HscC (Hsc62), homologue of Hsp70 in Escherichia coli: over‐expression of HscC modulates the activity of house keeping sigma factor σ70
Authors: Arifuzzaman Mohammad , Oshima Taku , Nakade Shinsuke , Mori Hirotada
Published: March 2003

Background: HscC, the third member of the Hsp70 family in Escherichia coli, shares 33% identity with the other two homologues, DnaK and HscA, and displays ATPase activity. Genetic and biochemical evidence indicates that the DnaK‐DnaJ chaperone system interacts with σ32 and is involved in the negative regulation of the heat shock response. Although HscC is a highly conserved protein in the Hsp70 family, its function is still unknown.

Results: We observed that the over‐expression of HscC caused severe growth inhibition. To explore this effect, we performed primer extension analysis and a β‐galactosidase assay and found that HscC reduced the σ70‐dependent promoter activity. An in vitro transcription assay revealed that HscC inhibited σ70‐dependent transcription. In addition, the co‐purification analysis showed that σ70 co‐eluted with HscC.

Conclusion: These results indicate that HscC forms a complex with σ70 and may function as its negative modulator.
Calcineurin phosphatase in signal transduction: lessons from fission yeast
Authors: Sugiura Reiko , Sio Susie O. , Shuntoh Hisato , Kuno Takayoshi
Published: March 2003

Calcineurin (protein phosphatase 2B), the only serine/threonine phosphatase under the control of Ca2+/calmodulin, is an important mediator in signal transmission, connecting the Ca2+‐dependent signalling to a wide variety of cellular responses. Furthermore, calcineurin is specifically inhibited by the immunosuppressant drugs cyclosporin A and tacrolimus (FK506), and these drugs have been a powerful tool for identifying many of the roles of calcineurin. Calcineurin is enriched in the neural tissues, and also distributes broadly in other tissues. The structure of the protein is highly conserved from yeast to man. The combined use of powerful genetics and of specific calcineurin inhibitors in fission yeast Schizosaccharomyces pombe (S. pombe) identified new components of the calcineurin pathway, and defined new roles of calcineurin in the regulation of the many cellular processes. Recent data has revealed functional interactions in which calcineurin phosphatase is involved, such as the cross‐talk between the Pmk1 MAP kinase signalling, or the PI signalling. Calcineurin also participates in membrane traffic and cytokinesis of fission yeast through its functional connection with members of the small GTPase Rab/Ypt family, and Type II myosin, respectively. These findings highlight the potential of fission yeast genetic studies to elucidate conserved elements of signal transduction cascades.