bluuebunny/crossref_metadata_2025 huggingface dataset:
Interactions between endangered wild and hatchery salmonids: can the pitfalls of artificial propagation be avoided in small coastal streams?
Authors: Hayes S. A. , Bond M. H. , Hanson C. V. , MacFarlane R. B.
Published: December 2004

Hatchery and wild juvenile populations of steelhead Oncorhynchus mykiss and coho salmon Oncorhynchus kisutch, in a small coastal watershed in central California, were sampled throughout the year in a stream and at a hatchery. Both species grew faster in captivity than in the wild. Hatchery fish of both species had elevated gill Na+, K+‐ATPase activity, and thus were ready to enter sea water when planted during the wild fish migration. Downstream migrant trapping and stream surveys indicated that hatchery smolts went to sea soon after planting, consequently avoiding the effects of competition and predation that commonly occur when hatchery‐bred juveniles are released. Adult steelhead were also sampled throughout the watershed. The return of hatchery steelhead was highly synchronized with that of wild steelhead, indicating that hatchery propagation had no adverse effects on the timing of the run. A disproportionate number of hatchery steelhead returned to the tributary where the hatchery was located, despite being planted throughout the watershed. Hatchery steelhead did not differ in mean age or size from wild steelhead. Observations of spawning indicated that hatchery and wild steelhead interbreed. Competition for mates or spawning substratum was rarely observed between hatchery and wild steelhead. Many of the problems commonly associated with artificial propagation can be avoided in small coastal watersheds when wild broodstock are used and fish are released as smolts.
A short hatchery history: does it make a difference to aggressiveness in European grayling?
Authors: Salonen A. , Peuhkuri N.
Published: December 2004

The level of aggressive behaviour in three populations of grayling Thymallus thymallus was lower in the hatchery strains than in the wild strains at the age of 0+ years. Due to similar rearing conditions, genetic divergence of the strains was most likely. As the hatchery fish used were second generation hatchery fish, this suggested that genetic changes in the hatchery can be very rapid. Therefore, it would be beneficial to use the progeny of wild fish for re‐introductions. Differences in aggressiveness between the strains still existed at the age of 1+ years, when the strains had been reared under common hatchery conditions for a year. A relatively short period in the hatchery may maintain the original behavioural characteristics of the fish and thus give the best possible basis for survival in the natural environment.
Life history responses of fishes to culture
Authors: Thorpe J. E.
Published: December 2004

The protected environment in culture permits fishes to reduce the proportion of energy normally channelled into the costs associated with competition for food, shelter and mates, avoidance of predators and counteracting parasites and diseases. The surplus energy so released is allocated to growth and reproduction, accelerating development through increased growth rate, earlier maturation and increased relative fecundity. Cultivators manipulate the rearing environment to remove seasonal variation in availability of resources, so that the fishes grow and develop through otherwise unproductive seasons. Such environmental manipulations exaggerate the basic accelerative effects. Since maturation deflects energy from growth, farmers also manipulate the fishes nutritionally, physiologically, hormonally and genetically to postpone maturation. As environmental regulators determine sex in many fish species, environmental manipulation in culture may have unintended effects on sex ratios. Mortality in culture should be very low, but survival of fishes released from culture is rarely as high as that of wild conspecifics. Finally, while short life‐cycles and simplified population age‐structures permit high rates of production in farms, they lead to ecological instability when the fishes are cultured for support of wild populations.
Stock enhancement and conservation culture of sturgeons: problems and prospects
Authors: Chebanov M. S. , Galich E. V. , Chmir Y. N.
Published: December 2004

Stock enhancement is the main source of Acipenser gueldenstaedtii and A. stellatus recruitment in the Sea of Azov, where up to 30 million juveniles have been released in recent years. The hatchery production strategy aimed solely to produce and release the largest possible number of juveniles, utilizing only the most mature broodfish of the spring spawning run. Although monitoring of age, mass and physiological conditions of the broodstock during the period of hatchery breeding did not show significant changes, the spawning run was dramatically shortened from several months to just 15 days, and includes only fish with advanced gonadal maturity. Conservation of the Azov Sea species and their unique spawning ecotypes is currently supported by the Federal Living Gene Bank, which maintains over 9000 adults of eight endangered species. This paper summarizes the results of comparative analysis of biological characteristics such as growth, age of the first sexual maturation, relative fecundity, and morphological and physiological indices in wild and cultured specimens of different species and intraspecific groups of sturgeons. We conclude that there is a need for developing better hatchery technologies to maintain diversity of the stocks, through breeding protocols that maximize the genetic diversity of offspring based on evaluation of parentage and relatedness in farmed stocks by microsatellite loci.
Reproductive ecology of cultured fish in the wild
Authors: Fleming I. A.
Published: December 2004

Fish show a tremendous diversity in patterns of reproductive investment and in associated breeding systems (i.e. parental care and sexual selection, including the number of mates obtained by both sexes and the manner in which they are obtained through competition for mates and resources, courtship, and mate choice). These patterns play an integral role in shaping the evolution of populations and their dynamics, and thus changes in these patterns necessarily affect population viability. Artificial culture of fish in hatcheries, net‐pens and gene banks almost invariably disrupts the natural breeding system and alters fitness‐related traits. The implications, both genetic and ecological, of the intentional and unintentional release of these fish for wild populations are largely dependent on what occurs during breeding and its subsequent effects on offspring performance. Our findings and those of others have indicated that gene flow from cultured to wild populations is frequently impeded by altered breeding behaviour and biased by sex and life history strategy. Moreover, breeding affects subsequent offspring performance through not only genetic (e.g., disruption of co‐adapted gene complexes, MHC non‐assortative mating), but also non‐genetic maternal effects (e.g., breeding time and location, egg size). While significant advances have been made in the last decade, our understanding of the reproductive ecology of cultured fish in the wild remains somewhat in its infancy. Such study continues to be integral in enlightening our management of cultured fishes in the wild, and more broadly for increasing our understanding of fish breeding systems and thus population dynamics.
Why do salmonid antipredator responses weaken in hatchery rearing?
Authors: Hirvonen H. , Laakkonen M. V. M.
Published: December 2004

The Arctic charr of Lake Saimaa are the most endangered fish population in Finland, and reintroduction programs have been unsuccessful. Low success of reintroduction programs has drawn attention to behavioural properties of hatcheryreared fish. Mortality due to predation often is a principal cause of failure. Antipredator behaviour may degenerate rapidly under hatchery conditions due to (i) reduced genetic variation in antipredator behaviour and/or (ii) selection that would favour bold and fast growing individuals and disfavour predator awareness supposedly associated with slow growth. To test the relative importance of these two factors we first analysed the amount of variation in innate antipredator responses between and within families of hatchery‐bred Arctic charr of the Lake Saimaa stock. We then tested whether fast growing individuals would show reduced responses to chemical cues from their natural predators compared to their slow growing counterparts. Based on the results we propose procedures for maintaining and improving antipredator skills of hatchery‐reared salmonids.
Disease interaction between farmed and wild fish populations
Authors: Peeler E. J. , Murray A. G.
Published: December 2004

This paper reviews the literature on disease interaction between wild and farmed fish and recommends strategies to reduce the disease risks to both populations. Most, if not all, diseases of farmed fish originate in wild populations. The close contact between farmed and wild fish readily leads to pathogens exchange. Aquaculture creates conditions (e.g. high stocking levels) conducive to pathogen transmission and disease; hence pathogens can overspill back, resulting in high levels of challenge to wild populations. This is exemplified by sea lice infections in farmed Atlantic salmon. Stocking with hatchery reared fish or aquaculture escapees can affect disease dynamics in wild populations. Whirling disease has been spread to many wild rainbow trout populations in the US with the release of hatchery reared stock. The greatest impact of aquaculture on disease in wild populations has resulted from the movement of fish for cultivation. Examples of exotic disease introduction following movement of live fish for aquaculture with serious consequences for wild populations are reviewed. The salmon parasite, Gyrodactylus salaris, has destroyed wild salmon populations in 44 Norwegian rivers. Crayfish plague has wiped out European crayfish over much of Europe. Eels numbers have declined in Europe and infection with the swimbladder nematode Anguillicola crassus has in part been blamed. The impact of disease in farmed fish on wild populations can mitigated. Risk analysis methods need to be refined and applied to live fish movement and new aquacultural developments. Appropriate biosecurity strategies, based on risk assessments, should be developed to reduce pathogen exchange and mitigate the consequences.
Life‐history traits of the common pandora Pagellus erythrinus L., interpreted using information from aquaculture experiments
Authors: Spedicato M. T. , Carbonara P. , Lembo G.
Published: December 2004

The common pandora Pagellus eythrinus is considered a promising species for Mediterranean aquaculture, due to its high market price and good reproduction in captive conditions. The aim of this paper is to clarify aspects of reproduction and growth in the common pandora, using data from both aquaculture studies and experimental fishing. Spawning time and behaviour, frequency of egg deposition, larval performance, growth of juveniles and adults and morphometric characteristics were studied in fish originating from a broodstock acclimatised to captivity since 1997. At the same time observations on length structure and other morphometric measures, age and patterns of sexual maturity were gathered from ten trawl‐surveys between 1996 and 2002. The surveys were carried out in late spring and early autumn in the Central‐Southern Tyrrhenian Sea, area where the breeders had been captured. Wild fish growth patterns were estimated using length frequency distribution analysis and age‐based methods. The study showed that several micro‐cohorts enter in the fishery over an extended period, and growth is faster than previously reported.
State and role of active conservation of barbel Barbus barbus in Poland
Authors: Kaczkowski Z. , Miroslaw C.
Published: December 2004

Barbel Barbus barbus is a highly valued target of recreational fisheries. Due to environmental degradation and growing angler pressure, its abundance has decreased in many rivers. In response to this, active protection strategies are being developed by scientists and the Polish Anglers Association. Methods of breeding in captivity have been developed for restocking purposes. Production and release have increased from 19 600 summer fry and 692 adult fish in 1998 to 7000 summer and 32 000 autumn juveniles, and 3150 two year‐old fish in 2002. The preservation of genetic diversity is a major challenge in these enhancement programmes.
Gonadal maturation in the blackspot seabream Pagellus bogaraveo: a comparison between a farmed and a wild broodstock
Authors: Micale V. , Maricchiolo G. , Genovese L.
Published: December 2004

The blackspot seabream Pagellus bogaraveo(Brünnich, 1768) has been regarded as a possible alternative to traditionally cultured Mediterranean species such as seabream and seabass, due to its high market value and good adaptation to captivity. Broodstock establishment and management represent the first step towards reliable production of eggs and fry, which is required to develop aquaculture of this new species. Two different broodstocks were tested for gonadal maturation and spawning, one constituting of wild fish caught as juveniles and reared in tanks until sexual maturity (4 years), and one assembled from wild adult fish caught during or just before the reproductive season. All fish were maintained under the same rearing conditions and fed the same diet. Gonadal stripping and biopsies were performed weekly to monitor maturation in both males and females. Ovarian samples were staged for maturity on the basis of follicular diameter and migration of germinal vesicle. Sperm samples were tested for density (number of spermatozoa ml−1) and motility. The fish reared in captivity reached ovarian maturity during the breeding season of the wild stock. Eggs were obtained by stripping from both farmed and wild specimens, but appeared degenerated as a result of being retained too long in the ovarian cavity due to the absence of spontaneous spawning. Spermiation was prolonged in the farmed fish, but appeared to be blocked in the wild breeders after first sampling. However, the sperm was very viscous and the motile spermatozoa did not exceed 10%.
Intra‐Firm Wage Dispersion and Firm Performance: Evidence from Linked Employer‐Employee Data
Authors: Lallemand Thierry , Plasman Robert , Rycx François
Published: December 2004

This paper examines the relationship between intra‐firm wage dispersion and firm performance in large Belgian firms using a unique matched employer‐employee data set. On the basis of the Winter‐Ebmer and Zweimüller's (1999) methodology, we find a positive and significant relationship between intra‐firm wage dispersion and profits per capita, even when controlling for individual and firm characteristics and addressing potential simultaneity problems. Results also suggest that the intensity of this relationship is stronger for blue‐collar workers and within firms with a high degree of monitoring. These findings are more in line with the ‘tournament’ models than with the ‘fairness, morale and cohesiveness’ models.
Convergence in Government Spending: Theory and Cross‐Country Evidence
Authors: Skidmore Mark , Toya Hideki , Merriman David
Published: December 2004

Under reasonable assumptions about production technologies, international competition for factors of production will lead to convergence of per capita output. Is there an analogous process that leads to convergence of public sector activity? We provide a simple model that predicts convergence in government spending under certain assumptions. We show that Barro's (1990) model of endogenous growth with government spending provides one justification for the necessary assumptions and therefore supports the convergence hypothesis. We also discuss the possibility that increasing globalization has led to convergence in government spending. Our cross‐country empirical investigation provides compelling evidence of convergence in per capita government consumption spending, per capita government capital spending, and per capita government education spending. These findings provide a new framework for explaining the underlying dynamic forces that determine growth of government.
Interpreting the World of Political Elites
Authors: Richards David , Smith Martin J.
Published: December 2004

This paper uses an interpretative approach to analyse the relationship between ministers and officials. It argues that generally, the relationship between ministers and civil servants is harmonious. This can be explained by the fact that both sets of actors tend to draw from the same tradition, the Westminster model. The Westminster model can be understood as the building block from which both ministers and civil servants develop narratives that shape and condition their actions. In the case of ministers, the dominant narrative drawn from the Westminster model is what we refer to as that of ‘historical impact’. In the case of civil servants, their dominant narrative, again drawn from the Westminster model is conditioned by the need to provide an account that continually emphasizes how they have acted with ‘constitutional propriety’. Generally, these two contrasting narratives do not lead to conflict between ministers and civil servants. However, a serious breakdown in the relationship between ministers and civil servants can occur when either one or both sets of actors draw on a tradition other than the Westminster model, or when they appeal to a different narrative within the Westminster model which shapes their subsequent behaviour and actions.
Local Government Reforms in Latvia, 1990–2003: Transition to a Democratic Society
Authors: King Gundar J. , Vanags Edvins , Vilka Inga , McNabb David E.
Published: December 2004

This case study of Latvian local governments focuses on the qualitative aspects of reform legislation passed and implemented between 1990 and 2003 as the best available measures of changes made. Special attention is given to historical influences and inherited Soviet practices, the uncertain and conflicting value systems of reformers, and a gradual shift to concerns about the services provided. Power relationships are examined, together with the funding of local governments and minority relations. It is anticipated that in 2005 Latvia will adopt new elected regional development boards to work with the EU and other external institutions, leading to more democratic and cooperative modes of government. There is evidence that values of achievement and trust held by the younger generation are increasingly compatible with new needs and reform processes.
Job‐Seeker Reactions to Selection Process Information in Job Ads
Authors: Reeve Charlie L. , Schultz Lisa
Published: December 2004

Using a policy‐capturing methodology, the current study examines if and to what extent individuals utilize selection process information contained in job ads in making evaluations of organizational attractiveness and decisions to apply. Results show both indirect and direct evidence that individuals do attend to and use these cues when presented in job ads in making initial job‐pursuit evaluations. Additionally, the results are consistent with a model derived from the organizational justice literature suggesting that perceptions of selection procedures' measurement accuracy may drive the observed effects. Specifically, ratings of organizational attractiveness and intentions to apply were significantly associated with perceptions of a given selection method's assessment accuracy for those ads that specified its use. Policy‐capturing results also show direct evidence that participants attend to and rely on selection process information in job ads to make job‐pursuit evaluations, and that perceptions of measurement accuracy are related to the degree to which, and how those cues are utilized. Research and applied implications are discussed.
Physiological and behavioural differences of hatchery and wild‐reared steelhead Oncorhynchus mykiss smolts of the same genetic origin
Authors: Gale W. L. , Hill M. S. , Zydlewski G. B.
Published: December 2004

In many parts of the world release of hatchery‐reared smolts has long been used to mitigate for the deleterious effects of habitat loss and overfishing on salmonid populations. Of increasing concern is whether this may cause harm by spreading non‐native stocks and potentially releasing incompetent smolts. The objective of this study was to determine if smolt physiology and behavior of juveniles produced from a recently founded native broodstock differ from their wild (naturally‐reared) counterparts. In the fall of 2002 and 2003 juvenile wild steelhead were captured, PIT tagged, and returned (n = 1360 in 2002 and n = 2708 in 2003) to Abernathy Creek. In winter of 2003 and 2004 hatchery‐reared fish were PIT tagged and later released (n = 1100 in 2003 and n = 1400 in 2004) into Abernathy Creek. Gill biopsies were collected from wild and hatchery fish throughout the rearing and out‐migration season. The timing and speed of outmigration was assessed using two stationary PIT tag antennas (92–97% efficient). Hatchery migrants in 2003 were larger, had significantly lower gill Na+, K+‐ATPase activities, and migrated slower than wild fish. Results from the 2004 migratory season will also be presented. This study shows that hatchery rearing can result in smolts which are physiologically and behaviourally different from genetically similar wild fish. Whether these differences are critical enough to affect the rate of adult returns will be determined in future years.
Bayesian estimation of cyclosporin exposure for routine therapeutic drug monitoring in kidney transplant patients
Authors: Bourgoin Hélène , Paintaud Gilles , Büchler Matthias , Lebranchu Yvon , Autret‐Leca Elisabeth , Mentré France , Guellec Chantal Le
Published: December 2004

Aims

AUC‐based monitoring of cyclosporin A (CsA) is useful to optimize dose adaptation in difficult cases. We developed a population pharmacokinetic model to describe dose‐exposure relationships for CsA in renal transplant patients and applied it to the Bayesian estimation of AUCs using three blood concentrations.

Methods

A total of 84 renal graft recipients treated with CsA microemulsion were included in this study. Population pharmacokinetic analysis was conducted using NONMEM. A two‐compartment model with zero‐order absorption and a lag time best described the data. Bayesian estimation was based on CsA blood concentrations measured before dosing and 1 h and 2 h post dose. Predictive performance was evaluated using a cross‐validation approach. Estimated AUCs were compared with AUCs calculated by the trapezoidal method. The Bayesian approach was also applied to an independent group of eight patients exhibiting unusual pharmacokinetic profiles.

Results

Mean population pharmacokinetic parameters were apparent clearance 30 l h−1, apparent volume of distribution 79.8 l, duration of absorption 52 min, absorption lag time 7 min. No significant relationships were found between any of the pharmacokinetic parameters and individual characteristics. A good correlation was obtained between Bayesian‐estimated and experimental AUCs, with a mean prediction error of 2.8% (95% CI [−0.6, 6.2]) and an accuracy of 13.1% (95% CI [7.5, 17.2]). A good correlation was also obtained in the eight patients with unusual pharmacokinetic profiles (r2 = 0.96, P < 0.01).

Conclusions

Our Bayesian approach enabled a good estimation of CsA exposure in a population of patients with variable pharmacokinetic profiles, showing its usefulness for routine AUC‐based therapeutic drug monitoring.
Purification, characterization and immunolocalization of porcine surfactant protein D
Authors: Soerensen C. M. , Nielsen O. L. , Willis A. , Heegaard P. M. H. , Holmskov U.
Published: December 2004

Surfactant protein D (SP‐D) is a collectin believed to play an important role in innate immunity. SP‐D is characterized by having a collagen‐like domain and a carbohydrate recognition domain (CRD), which has a specific Ca2+‐dependent specificity for saccharides and thus the ability to bind complex glycoconjugates on micro‐organisms. This paper describes the tissue immunolocalization of porcine SP‐D (pSP‐D) in normal slaughter pigs using a monoclonal antibody raised against purified pSP‐D. Porcine SP‐D was purified from porcine bronchoalveolar lavage (BAL) by maltose‐agarose and immunoglobulin M affinity chromatography. The purified protein appeared on sodium dodecyl sulphate–polyacrylamide gel electrophoresis as a band of ∼53 000 MW in the reduced state and ∼138 000 MW in the unreduced state. Porcine SP‐D was sensitive to collagenase digestion and N‐deglycosylation, which reduced the molecular mass to ∼24 000 MW and ∼48 000 MW respectively, in the reduced state. N‐deglycosylation of the collagen‐resistant fragment, reduced the molecular mass to ∼21 000 MW showing the presence of an N‐glycosylation site located in the CRD. Porcine SP‐D bound to solid‐phase mannan in a dose and Ca2+‐dependent manner with a saccharide specificity similar to rat and human SP‐D. The purified protein was used for the production of a monoclonal anti‐pSP‐D antibody. The antibody reacted specifically with pSP‐D in the reduced and unreduced state when analysed by Western blotting. Immunohistochemical evaluation of normal porcine tissues showed pSP‐D immunoreactivity predominantly in Clara cells and serous cells of the bronchial submucosal glands, and to a lesser extent in alveolar type II cells, epithelial cells of the intestinal glands (crypts of Lieberkühn) in the duodenum, jejunum and ileum and serous cells of the dorsolateral lacrimal gland.
Enhanced viral clearance in interleukin‐18 gene‐deficient mice after pulmonary infection with influenza A virus
Authors: Van Der Sluijs Koenraad F. , Van Elden Leontine J. R. , Arens Ramon , Nijhuis Monique , Schuurman Rob , Florquin Sandrine , Kwakkel Joan , Akira Shizuo , Jansen Henk M. , Lutter René , Van Der Polls Tom
Published: December 2004

T helper 1 driven immune responses facilitate host defence during viral infections. Because interleukin‐18 (IL‐18) mediates T helper 1 driven immune responses, and since mature IL‐18 is up‐regulated in human macrophages after influenza virus infection in vitro, it has been suggested that IL‐18 plays an important role in the immune response to influenza. To determine the role of IL‐18 in respiratory tract infection with influenza, IL‐18 gene‐deficient (IL‐18–/–) and normal wildtype mice were intranasally inoculated with influenza A virus. Influenza resulted in an increase in constitutively expressed IL‐18 in the lungs of wildtype mice. The clearance of influenza A was inhibited by IL‐18, as indicated by reduced viral loads on day 8 and day 12 after infection in IL‐18–/– mice. This enhanced viral clearance correlated with increased CD4+ T‐cell activation in the lungs as reflected by CD69 expression on the cell surface. Surprisingly, interferon‐γ (IFN‐γ) levels were similar in the lungs of IL‐18–/– mice and wildtype mice. Intracellular IFN‐γ staining revealed similar expression levels in lung‐derived natural killer cells, CD4+ and CD8+ T cells, indicating that IFN‐γ production is IL‐18‐independent during influenza virus infection. Tumour necrosis factor‐α production by CD4+ T cells was significantly lower in IL‐18–/– mice than in wildtype mice. Our data indicate that endogenous IL‐18 impairs viral clearance during influenza A infection.
The role of chemokines and extracellular matrix components in the migration of T lymphocytes into three‐dimensional substrata
Authors: Ivanoff Jyrki , Talme Toomas , Sundqvist Karl‐Gösta
Published: December 2004

The role of chemokines and their interactions with extracellular matrix components (ECM) or the capacity of T cells to migrate into and accumulate within three‐dimensional (3D) collagen type 1 substrata was studied. We examined the influence of chemokines and fibronectin on the infiltration properties of non‐infiltrative (do not migrate into 3D substrata) and spontaneously infiltrative (migrate into 3D substrata) T‐cell lines. Infiltrative and non‐infiltrative T‐acute lymphocytic leukaemic cell lines exhibited no consistent differences with respect to the expression of various chemokine receptors or β1‐integrins. Chemokines presented inside the collagen increased the depth of migration of infiltrative T‐cell lines, but did not render non‐infiltrative T‐cell lines infiltrative, although they augmented the attachment of non‐infiltrative T‐cell lines to the upper surface of the collagen. The presence of fibronectin inside the collagen did not render non‐infiltrative T‐cell lines infiltrative, but markedly augmented the migration of ‘infiltrative’ T‐cell lines into collagen. Both infiltrative and non‐infiltrative T‐cell lines showed migratory responses to chemokines in Boyden assays (migration detected on 2D substrata). These results indicate that the process of T‐cell infiltration/migration into 3D substrata depends on a tissue penetration mechanism distinguishable from migration on 2D substrata and that the basic capacity of T cells to infiltrate is independent of chemokines and ECM components applied as attractants.
Age‐related loss of naïve T cells and dysregulation of T‐cell/B‐cell interactions in human lymph nodes
Authors: Lazuardi Lutfan , Jenewein Brigitte , Wolf Anna Maria , Pfister Gerald , Tzankov Alexandar , Grubeck‐Loebenstein Beatrix
Published: December 2004

In this study we analysed the effects of age on T and B lymphocytes in human lymph nodes by comparing lymphocyte subsets in paraffin sections from lymph node tissue taken from healthy young and elderly people. We demonstrate that the relative number of CD8+ T cells decreases with age but that the relative number of CD4+ T cells does not. There is also a very pronounced age‐dependent loss of CD45RA+ naïve T cells. The number and size of follicles and the relative number of CD20+ B cells are similar in young and elderly donors. For polymerase chain reaction analysis of the T‐cell receptor (TCR) repertoire the TCR‐γ gene rearrangements were used as a marker of clonality. This is a reliable tool to detect not only clonal TCR‐γδ populations but also TCR‐αβ populations. Young donors with clonal T‐cell expansions in their lymph node tissue do, however, have a higher number of CD20+ B cells, a higher relative size of germinal centres compared to the follicle mantles and a higher number of immunoglobulin M‐expressing cells than young donors without evidence of clonal T‐cell expansions. Corresponding changes are not observed in elderly donors with clonal T‐cell expansions in their lymph node tissue. In summary our findings demonstrate characteristic effects of aging on human lymph node tissue, the most striking feature being the depletion of naïve T cells and the apparent dysregulation of T‐cell/B‐cell interactions in old age.
Analysis of the proteins targeted by CDSP32, a plastidic thioredoxin participating in oxidative stress responses
Authors: Rey Pascal , Cuiné Stéphan , Eymery Françoise , Garin Jérome , Court Magali , Jacquot Jean‐Pierre , Rouhier Nicolas , Broin Mélanie
Published: December 2004

The chloroplastic drought‐induced stress protein of 32 kDa (CDSP32) is a thioredoxin induced by environmental stress conditions. To gain insight into the function of CDSP32, we applied two strategies to analyze its targets. First, using affinity chromatography with an immobilized CDSP32 active site mutant, we identified six plastidic targets of CDSP32. Three of them are involved in photosynthetic processes: ATP‐aseγ‐subunit, Rubisco and aldolase. The three others participate in the protection against oxidative damage: two peroxiredoxins, PrxQ and the BAS1 2‐Cys peroxiredoxin, and a B‐type methionine sulfoxide reductase. Then, we developed a novel strategy to trap targets directly in leaf extracts. The method, based on co‐immunoprecipitation using extracts from plants overexpressing Wt CDSP32 or CDSP32 active site mutant, confirmed the interactionin vivobetween CDSP32 and the PrxQ and BAS1 peroxiredoxins. We showed that CDSP32 is able to form heterodimeric complexes with PrxQ and that the peroxiredoxin displays CDSP32‐dependent peroxidase activity. Under photooxidative stress induced by methyl viologen, plants overexpressing CDSP32 active site mutant exhibit decreased maximal PSII photochemical efficiency and retain much less chlorophyll compared with Wt plants and with plants overexpressing Wt CDSP32. We propose that the increased sensitivity results from trappingin plantaof the targets involved in the protection against oxidative damage. We conclude that CDSP32, compared with other plant thioredoxins, is a thioredoxin more specifically involved in plastidic responses against oxidative stress.
Abscisic acid and 14‐3‐3 proteins control K+ channel activity in barley embryonic root
Authors: van den Wijngaard Paul W.J. , Sinnige Mark P. , Roobeek Ilja , Reumer Annet , Schoonheim Peter J. , Mol Jos N.M. , Wang Mei , De Boer Albertus H.
Published: December 2004

Germination of seeds proceeds in general in two phases, an initial imbibition phase and a subsequent growth phase. In grasses like barley, the latter phase is evident as the emergence of the embryonic root (radicle). The hormone abscisic acid (ABA) inhibits germination because it prevents the embryo from entering and completing the growth phase. Genetic and physiological studies have identified many steps in the ABA signal transduction cascade, but how it prevents radicle elongation is still not clear. For elongation growth to proceed, uptake of osmotically active substances (mainly K+) is essential. Therefore, we have addressed the question of how the activity of K+ permeable ion channels in the plasma membrane of radicle cells is regulated under conditions of slow (+ABA) and rapid germination (+fusicoccin). We found that ABA arrests radicle growth, inhibits net K+ uptake and reduces the activity of K+in channels as measured with the patch‐clamp technique. In contrast, fusicoccin (FC), a well‐known stimulator of germination, stimulates radicle growth, net K+ uptake and reduces the activity of K+out channels. Both types of channels are under the control of 14‐3‐3 proteins, known as integral components of signal transduction pathways and instrumental in FC action. Intriguingly, 14‐3‐3 affected both channels in an opposite fashion: whereas K+in channel activity was fully dependent upon 14‐3‐3 proteins, K+out channel activity was reduced by 14‐3‐3 proteins by 60%. Together with previous data showing that 14‐3‐3 proteins control the activity of the plasma membrane H+‐ATPase, this makes 14‐3‐3 a prime candidate for molecular master regulator of the cellular osmo‐pump. Regulation of the osmo‐pump activity by ABA and FC is an important mechanism in controlling the growth of the embryonic root during seed germination.
Functional genomics uncovers three glucosyltransferases involved in the synthesis of the major sweet glucosides of Stevia rebaudiana
Authors: Richman Alex , Swanson Andrew , Humphrey Tania , Chapman Ralph , McGarvey Brian , Pocs Robert , Brandle Jim
Published: December 2004

Stevia rebaudiana leaves accumulate a mixture of at least eight different steviol glycosides. The pattern of glycosylation heavily influences the taste perception of these intensely sweet compounds. The majority of the glycosides are formed by four glucosylation reactions that start with steviol and end with rebaudioside A. The steps involve the addition of glucose to the C‐13 hydroxyl of steviol, the transfer of glucose to the C‐2′ and C‐3′ of the 13‐O‐glucose and the addition of glucose to the hydroxyl of the C‐4 carboxyl group. We used our collection of ESTs, an UDP‐glucosyltransferase (UGT)‐specific electronic probe and key word searches to identify candidate genes resident in our collection. Fifty‐four expressed sequence tags (ESTs) belonging to 17 clusters were found using this procedure. We isolated full length cDNAs for 12 of the UGTs, cloned them into an expression vector, and produced recombinant enzymes in Escherichia coli. An in vitro glucosyltransferase activity enzyme assay was conducted using quercetin, kaempferol, steviol, steviolmonoside, steviolbioside, and stevioside as sugar acceptors, and 14C‐UDP‐glucose as the donor. Thin layer chromatography was used to separate the products and three of the recombinant enzymes produced labelled products that co‐migrated with known standards. HPLC and LC‐ES/MS were then used to further define those reaction products. We determined that steviol UGTs behave in a regioselective manner and propose a modified pathway for the synthesis of rebaudioside A from steviol.
Identification of translocatable RNA‐binding phloem proteins from melon, potential components of the long‐distance RNA transport system
Authors: Gómez G. , Torres H. , Pallás V.
Published: December 2004

Phloem proteins (P‐proteins) are an enigmatic group of proteins present in most angiosperm species. The best characterized P‐proteins (PP1 and PP2) are synthesized in companion cells, transported into sieve elements via pore plasmodesmata and translocated through the plant. Characteristics such as long‐distance translocation, RNA‐binding activity and capacity of increasing plasmodesmata exclusion size suggest that certain phloem proteins could be involved in RNA transport within the plant, forming translocatable ribonucleoprotein complexes with endogenous or pathogenic RNAs. Long‐distance movement of RNA through the phloem is a process known to occur, but both the mechanisms involved and the components constituting this potential information network remain unclear. Here, we demonstrate that several melon phloem proteins have a wide RNA‐binding activity. Serological assays strongly suggest that one of these proteins is the melon phloem protein 2 (CmmPP2). Mass spectrometry analysis undoubtedly identifies another one as the recently characterized melon phloem lectin (CmmLec17). Grafting experiments demonstrate that the CmmLec17 is a translocatable phloem protein, able to move through intergeneric grafts from melon to pumpkin. Translocatability and RNA‐binding activity was also demonstrated for an uncharacterized protein of approximately 14 kDa. In light of these results the possible involvement of these phloem proteins in the long‐distance transport of melon RNAs is discussed.
Membrane‐anchored prolyl hydroxylase with an export signal from the endoplasmic reticulum
Authors: Yuasa Koji , Toyooka Kiminori , Fukuda Hiroo , Matsuoka Ken
Published: December 2004

We cloned a novel prolyl 4‐hydroxylase (PH; EC 1.14.11.2) homolog cDNA from tobacco (Nicotiana tabacum) BY‐2 cells based on expression sequence tag information. Like other PHs, this tobacco PH polypeptide has two conserved histidine residues, and it comprises 286 amino acids with a calculated molecular mass of 32 kDa. Interestingly, this protein and homologs in Arabidopsis and rice have predicted transmembrane sequences in their N‐terminal regions. This PH homolog was expressed in BY‐2 cells as a His‐tagged protein, and the expressed protein showed PH activity. Incubation of membranes with high salt, urea, and protease with or without detergents indicated that this protein is an integral membrane protein with a type II configuration. Its membrane‐anchored nature is specific for plants because no integral membrane PH has been found in animals. A membrane fractionation study and immunocytochemical studies indicate that this protein localizes in both the endoplasmic reticulum (ER) and Golgi apparatus. Analysis of this protein fused to green fluorescent protein indicated that basic amino acids in the cytoplasmic, N‐terminal region of the PH play a role in its export from the ER.
Arabidopsis vacuolar H+‐ATPase subunit E isoform 1 is required for Golgi organization and vacuole function in embryogenesis
Authors: Strompen Georg , Dettmer Jan , Stierhof York‐Dieter , Schumacher Karin , Jürgens Gerd , Mayer Ulrike
Published: December 2004

Vacuolar H+‐ATPases play an important role in maintaining the pH of endomembrane compartments in eukaryotic cells. The functional relevance of this homeostasis for multicellular development has not been studied in plants. Here, we analyze the biological consequences resulting from the lack of subunit E isoform 1 (VHA‐E1) encoded by the Arabidopsis TUFF gene. tuff mutant embryos are lethal, displaying variably enlarged cells with multiple nuclei, large vacuoles containing inclusions, abnormal organization of Golgi stacks, and cell wall defects. Rescue of embryo lethality by cell cycle‐regulated expression of VHA‐E1 results in abnormal seedlings with non‐functional meristems and defective cell differentiation. VHA‐E1 is the predominant isoform in embryogenesis whereas VHA‐E3 is expressed mainly in the endosperm and surrounding maternal tissues during seed development, and VHA‐E2 is pollen‐specific. VHA‐E1 protein accumulates at endomembrane compartments including vacuoles and endosomes, but appears absent from the plasma membrane. Our results suggest an essential role for VHA‐E1 in maintaining a functional secretory system during somatic development but not in the haploid gametophytes.
β‐1,3‐Glucanase gene expression in low‐hydrated seeds as a mechanism for dormancy release during tobacco after‐ripening
Authors: Leubner‐Metzger Gerhard
Published: December 2004

An air‐dry developmental state with low‐hydrated tissues is a characteristic of most plant seeds. Seed dormancy is an intrinsic block of germination and can be released during after‐ripening, that is air‐dry storage of mature seeds. Both seed‐covering layers, testa and endosperm, cause the coat‐imposed dormancy of tobacco (Nicotiana tabacum). After‐ripening and over‐expression of class I β‐1,3‐glucanase (βGlu I) confer maternal effects on testa rupture and dormancy release. Very little is known about the molecular mechanisms of after‐ripening and whether gene expression is possible in low‐hydrated seeds. Transient, low‐level βGlu I transcription and translation was detected during tobacco seed after‐ripening. 1H NMR 2D micro‐imaging showed uneven distribution of proton mobility in seeds. βGlu I gene expression is associated spatially with the inner testa and temporally with the promotion of testa rupture. Local elevation in moisture content seems to permit local, low‐level βGlu I gene transcription and translation in the maternal tissues of air‐dry, low‐hydrated seeds. De novo gene expression is therefore proposed to be a novel molecular mechanism for the release of coat‐imposed dormancy during oilseed after‐ripening.
A new type of mutation in phytochrome A causes enhanced light sensitivity and alters the degradation and subcellular partitioning of the photoreceptor
Authors: Dieterle Monika , Bauer Diana , Büche Claudia , Krenz Martina , Schäfer Eberhard , Kretsch Thomas
Published: December 2004

A specific light program consisting of multiple treatments with alternating red and far‐red light pulses was used to isolate mutants in phytochrome A‐dependent signal transduction pathways in Arabidopsis. Because of their phenotype, the mutants were called eid for empfindlicher im dunkelroten Licht, which means hypersensitive in far‐red light. One of the isolated mutants, eid4, is a novel semi‐dominant allele of the phytochrome A gene that carries a missense mutation in the chromophore‐binding domain. The mutation did not change the photochemical properties of the photoreceptor, but it leads to an increased stability under light conditions that induce its rapid degradation. Fusion proteins with the green fluorescent protein exhibited clear alterations in subcellular localization of the mutated photoreceptor: The fusion protein was impaired in the formation of sequestered areas of phytochrome in the cytosol, which can explain its reduced light‐dependent degradation. In contrast, the mutation stabilizes nuclear speckles (NUS) that appear late under continuous far‐red light, whereas the formation of early, transiently appearing NUS remained more or less unaltered.
Altered expression of CD1d molecules and lipid accumulation in the human hepatoma cell line HepG2 after iron loading
Authors: Cabrita Marisa , Pereira Carlos F. , Rodrigues Pedro , Cardoso Elsa M. , Arosa Fernando A.
Published: December 2004

Iron overload in the liver may occur in clinical conditions such as hemochromatosis and nonalcoholic steatohepatitis, and may lead to the deterioration of the normal liver architecture by mechanisms not well understood. Although a relationship between the expression of ICAM‐1, and classical major histocompatibility complex (MHC) class I molecules, and iron overload has been reported, no relationship has been identified between iron overload and the expression of unconventional MHC class I molecules. Herein, we report that parameters of iron metabolism were regulated in a coordinated‐fashion in a human hepatoma cell line (HepG2 cells) after iron loading, leading to increased cellular oxidative stress and growth retardation. Iron loading of HepG2 cells resulted in increased expression of Nor3.2‐reactive CD1d molecules at the plasma membrane. Expression of classical MHC class I and II molecules, ICAM‐1 and the epithelial CD8 ligand, gp180 was not significantly affected by iron. Considering that intracellular lipids regulate expression of CD1d at the cell surface, we examined parameters of lipid metabolism in iron‐loaded HepG2 cells. Interestingly, increased expression of CD1d molecules by iron‐loaded HepG2 cells was associated with increased phosphatidylserine expression in the outer leaflet of the plasma membrane and the presence of many intracellular lipid droplets. These data describe a new relationship between iron loading, lipid accumulation and altered expression of CD1d, an unconventional MHC class I molecule reported to monitor intracellular and plasma membrane lipid metabolism, in the human hepatoma cell line HepG2.
Gain of structure and IgE epitopes by eukaryotic expression of the major Timothy grass pollen allergen, Phl p 1
Authors: Ball Tanja , Edstrom William , Mauch Ludwig , Schmitt Jacky , Leistler Bernd , Fiebig Helmut , Sperr Wolfgang R. , Hauswirth Alexander W. , Valent Peter , Kraft Dietrich , Almo Steven C. , Valenta Rudolf
Published: December 2004

Approximately 400 million allergic patients are sensitized against group 1 grass pollen allergens, a family of highly cross‐reactive allergens present in all grass species. We report the eukaryotic expression of the group 1 allergen from Timothy grass, Phl p 1, in baculovirus‐infected insect cells. Domain elucidation by limited proteolysis and mass spectrometry of the purified recombinant glycoprotein indicates that the C‐terminal 40% of Phl p 1, a major IgE‐reactive segment, represents a stable domain. This domain also exhibits a significant sequence identity of 43% with the family of immunoglobulin domain‐like group 2/3 grass pollen allergens. Circular dichroism analysis demonstrates that insect cell‐expressed rPhl p 1 is a folded species with significant secondary structure. This material is well behaved and is adequate for the growth of crystals that diffract to 2.9 Å resolution. The importance of conformational epitopes for IgE recognition of Phl p 1 is demonstrated by the superior IgE recognition of insect‐cell expressed Phl p 1 compared to Escherichia coli‐expressed Phl p 1. Moreover, insect cell‐expressed Phl p 1 induces potent histamine release and leads to strong up‐regulation of CD203c in basophils from grass pollen allergic patients. Deglycosylated Phl p 1 frequently exhibits higher IgE binding capacity than the recombinant glycoprotein suggesting that rather the intact protein structure than carbohydrate moieties themselves are important for IgE recognition of Phl p 1. This study emphasizes the important contribution of conformational epitopes for the IgE recognition of respiratory allergens and provides a paradigmatic tool for the structural analysis of the IgE allergen interaction.